Application of saikoside D as inducer of unfolded protein reaction in prostatic cancer

Abstract

The invention discloses application of saikoside D as an inducer of unfolded protein reaction in prostatic cancer, and belongs to the technical field of biological medicine. The invention finds for the first time that SSD activates UPR by promoting phosphorylation of 51-site Ser in EIF2alpha, causes the failure of a cancer cell endoplasmic reticulum sensor and the emergency situation that protein synthesis is obviously inhibited, and further causes cell death and inhibition of cancer cell proliferation. According to the SSD disclosed by the invention, the expression of Phospho-eIF2 alpha (Ser51) can be obviously activated, and it is clear that UPR is a potential target spot of the SSD. The SSD provided by the invention can more effectively inhibit proliferation of prostate cancer cells by enhancing UPR in prostate cancer cells and combining with a common drug Enzaluan for prostate cancer.

Description

Technical Field

[0001] The present invention belongs to biomedical technology, and particularly relates to the application of saikosaponin D as an inducer of unfolded protein response in prostate cancer. Background Art

[0002] Prostate cancer is one of the most common malignant tumors of the male urinary reproductive system. In clinical practice, castration therapy is often chosen for prostate cancer patients. Its purpose is to remove the production of testicular androgens through surgery or drugs so that the serum testosterone concentration reaches the castration level, which is less than 5%-10% of the baseline value. Although castration therapy is effective for most prostate cancer patients in the early stages, almost all patients will eventually develop castration-resistant prostate cancer (CRPC), at which time the cancer loses its response to castration therapy. The resistance mechanism of CRPC involves a variety of complex biological processes, including androgen receptor (AR) mutations, AR signaling pathway activation, etc., which makes it extremely difficult to develop new treatments for these mechanisms.

[0003] Protein homeostasis is an important component of cellular homeostasis related to prostate cancer metabolism. The integrated stress response (ISR) in cancer cells is activated when experiencing intrinsic or extrinsic damage. It is a conserved intracellular signal transduction network, and the unfolded protein response (UPR) is an adaptive response to ISR activation. During prostate tumorigenesis, the large demand for endoplasmic reticulum (ER) protein folding and limited capacity during cancer cell proliferation will lead to the accumulation of improperly folded proteins. Under general ER stress, the cell's own adjustment can regulate cell homeostasis and promote cell survival; however, under long-term ER stress, the dysregulation of UPR will cause the cell to be unable to restore cellular homeostasis and lead to its death. Common UPR inducers include: Thapsigargin, Tunicamycin, and Fumonisin B1.

[0004] Saikosaponin D (SSD), Cas number: 20874-52-6, is a type of saponin monomer component extracted from the dried roots of Bupleurum chinense and Bupleurum angustifolia of the Umbelliferae family. It is one of the main chemical indicators and bioactive components in Bupleurum chinense. Summary of the invention

[0005] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide the use of saikosaponin D in the preparation of an inducer of unfolded protein response.

[0006] Another object of the present invention is to provide the use of saikosaponin D as an inducer of unfolded protein response in the preparation of a drug for treating prostate cancer.

[0007] Another object of the present invention is to provide a use of saikosaponin D combined with Enzalutamide in the preparation of a drug for treating prostate cancer.

[0008] Saikosaponin D can cause the failure of endoplasmic reticulum sensors in prostate cancer cells, fully induce cancer cell death and inhibit cancer cell proliferation, and has potential therapeutic effects on prostate cancer patients.

[0009] The purpose of the present invention is achieved through the following technical solutions: The use of saikosaponin D in the preparation of an inducer of unfolded protein response, wherein the structural formula of saikosaponin D is shown in Formula 1: Formula 1.

[0010] Furthermore, the effective concentration of saikosaponin D is 1-50 μmol / L, and the preferred concentration range is 1-20 μmol / L.

[0011] Application of saikosaponin D as an inducer of unfolded protein response in the preparation of drugs for treating prostate cancer.

[0012] Furthermore, the effective concentration of saikosaponin D is 1-50 μmol / L, and the preferred concentration range is 1-20 μmol / L.

[0013] Application of saikosaponin D combined with Enzalutamide in the preparation of drugs for the treatment of prostate cancer.

[0014] Furthermore, the effective concentration of the saikosaponin D is 5 to 25 μmol / L; the effective concentration of Enzalutamide is 10 to 20 μmol / L.

[0015] Furthermore, the effective concentration of the saikosaponin D is 5 μmol / L; the effective concentration of Enzalutamide is 20 μmol / L.

[0016] Furthermore, the drug for treating prostate cancer also includes pharmacodynamically acceptable excipients.

[0017] Furthermore, the pharmaceutically acceptable excipient is preferably at least one of a sustained-release agent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, an adsorption carrier, a surfactant and a lubricant.

[0018] Furthermore, the administration methods of the drug for treating prostate cancer include but are not limited to oral administration and injection administration.

[0019] Compared with the prior art, the present invention has the following advantages and effects: The present invention discovered for the first time that saikosaponin D (SSD) can significantly induce UPR in cells, thereby causing the failure of endoplasmic reticulum sensors in cancer cells, further inhibiting the proliferation of prostate cancer cells, and can significantly improve the effect of promoting cancer cell death by combining with the clinical drug Enzalutamide.

[0020] The present invention confirms that saikosaponin D can significantly regulate the UPR pathway in tissue samples of prostate cancer patients.

[0021] The present invention confirms that saikosaponin D can significantly inhibit the proliferation of prostate cancer cells.

[0022] The present invention confirms that the combined use of Enzalutamide and other target drugs has significant clinical potential in the treatment of prostate cancer and can provide patients with more treatment options and better treatment effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Based on the TCGA public database, the sequencing results of prostate cancer tissue samples and non-cancerous tissue samples at the transcriptome level were compared to conduct GESA enrichment analysis at the gene level and significant enrichment maps of the UPR pathway; Figure 2 This is a statistical analysis chart showing that saikosaponin D can significantly inhibit the proliferation of prostate cancer cells and induce their death; Figure 3 This is a protein immunoblot image showing that saikosaponin D can promote the phosphorylation of Ser 51 in intracellular EIF2α to activate UPR; Figure 4 This is a statistical analysis chart showing that saikosaponin D can significantly activate UPR in prostate cancer cells LNCaP; Figure 5 This is the interaction diagram between saikosaponin D and the key protein molecule of p-EIF2α; Figure 6 This is a phase contrast image showing that saikosaponin D can combine with the clinical drug Enza to enhance the inhibition of cancer cell proliferation and induce its death; Figure 7 This is a statistical analysis chart showing that saikosaponin D can combine with the clinical drug Enza to enhance the inhibition of cancer cell proliferation and induce their death. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with the embodiments. The equipment and reagents used in each embodiment and test example can be obtained from commercial sources unless otherwise specified. The specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0025] The present invention obtains through in vitro experimental studies that saikosaponin D can significantly inhibit the proliferation of prostate cancer cells, and finds that it mainly inhibits the proliferation of cancer cells by fully activating the UPR in cells.

[0026] Example 1: Saikosaponin D can significantly inhibit the proliferation of prostate cancer cells and induce their death Experimental Materials: Cell line: LNCaP (human prostate cancer cell); Culture medium: Roswell Park Memorial Institute (RPMI) 1640 with 10% FBS (Fetal Bovine Serum), 100 U / ml penicillin and 0.1 mg / ml streptomycin; Trypsin: 0.25% trypsin-EDTA solution; Compound: saikosaponin D; Incucyte® Cytotox Green Dye: used to mark dead cells in culture; Incucyte real-time live cell imaging system: used to directly measure the cell confluence or cell number changes at different time points, and provide cell proliferation curves over time; Corning® 96-well clear flat-bottom polystyrene microplate: used for real-time cell analysis experiments; CO2 incubator: used to maintain the environment required for cell growth; Microscope and cell counting chamber: used for cell counting.

[0027] Experimental methods: Cell culture: LNCaP cells were inoculated into culture flasks containing RPMI 1640 medium and cultured at 37°C and 5% CO2. When the cells grew to 80%-90% confluence, they were digested with 0.25% trypsin and passaged.

[0028] Cell counting: Use 0.25% trypsin to digest the cells, wash with PBS (Phosphate Buffered Saline), and drop the cell suspension into a cell counting plate. Count the number of cells under a microscope and calculate the number of cells per milliliter.

[0029] Cell plating and proliferation experiment: LNCaP cells were plated at a density of 5000 / 100 μL / well in a Corning® 96-well plate containing appropriate culture medium and placed in an Incucyte incubator overnight. Sterile DMSO was used to prepare saikosaponin D into a 20 mmol / L drug stock solution. After 24 hours of plating, saikosaponin D with a concentration gradient of 0, 1, 5, 10, 15, and 20 μmol / L was added. The number of repetitions for each group was n=5, and it was fully ensured that the cells were continuously in a constant temperature environment during the experiment to observe the dynamic changes of the cells.

[0030] Experimental results: During the real-time cell monitoring process, the Incucyte 10X objective lens magnification was selected, and cell proliferation was automatically detected every 2 hours for long-term continuous monitoring. The confluence percentage of the phase contrast image was used as the cell coverage (Confluence) parameter value to evaluate the changes in cell proliferation. The cell proliferation curve was plotted using the software GraphPad Prism 8.0.

[0031] like Figure 2 As shown, the present invention confirms that LNCaP can cause cell death and loss of cell membrane integrity under the action of different concentrations of saikosaponin D, especially when the concentration is greater than 10 μmol / L, and Cytotox Green Dye dye enters the cell and fluorescently marks the cell nucleus, thereby being identified and quantified. The results show that saikosaponin D can successfully kill cells and inhibit the proliferation of tumor cells at a certain concentration. The experimental results p < 0.05 is *, indicating a statistical difference; p < 0.01 is **, indicating a significant difference; p < 0.001 is ***, indicating an extremely significant difference.

[0032] like Figure 3 As shown, the present invention confirms that saikosaponin D can significantly change cell morphology at a certain concentration, causing the cells to shrink and die, confirming that saikosaponin D does not act by inducing cell apoptosis, but may promote cell death through a certain stress response. The present invention proposes that it may be used as a UPR inducer in promoting cancer cell death by inducing endoplasmic reticulum stress.

[0033] Example 2: Saikosaponin D can fully induce the unfolded protein response in prostate cancer cells Experimental Materials Cell lines: LNCaP (human prostate cancer cells), 293T (human kidney epithelial cell line); Culture medium: Roswell Park Memorial Institute (RPMI) 1640 + 10% FBS (Fetal Bovine Serum) containing 100 U / ml penicillin and 0.1 mg / ml streptomycin; Dulbecco's Modified Eagle Medium (DMEM) + 10% FBS containing 100 U / ml penicillin and 0.1 mg / ml streptomycin; Enzyme: 0.25% trypsin-EDTA solution; Compound: Saikosaponin D, Thapsigargin; UPR probe plasmid: characterize the dynamic changes of UPR in cells (gifted by the State Key Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, published in the literature “Xu, Fenfen, and Likun Wang. “Deciphering ERstress-unfolded protein response relationship by visualizing unfolded proteins inthe ER.” Cell reports vol. 43,6 (2024): 114358.doi:10.1016 / j.celrep.2024.114358”); Plasmid extraction kit: used to extract the target plasmid; Lipofectamine 3000: Uses lipid nanoparticle technology to introduce exogenous DNA (plasmid) into the target cell LNCaP.

[0034] Experimental protocol: Extract the target plasmid UPR-GFP: Transform the UPR probe plasmid into E. coli, and then culture it in LB medium containing amp antibiotics. When the bacteria grow to an appropriate density (OD600 is about 0.6-1.2), collect the bacteria. Extract the target plasmid according to the instructions of the plasmid extraction kit and determine the plasmid DNA concentration.

[0035] Lentivirus packaging: Take out the logarithmically growing 293T cells from the incubator, coat the 6-well plate with 0.025 mg / mL poly-lysine at 37°C for 10 min, then rinse with PBS 3 times, discard the PBS and dry for later use, then inoculate the 293T cells in the above 6-well plate, and wait for the cell density to be greater than 80% and in good condition before preparing the cell transfection experiment. First, prepare a certain amount of 1.5 mL EP tubes, in which 750 μl (250 μl / well) of Opti-MEM medium is added to each of the two tubes (temporarily called A and B tubes), and 27 μl of lipo 3000 (DNA:lipo3000=1:2.5) is added to tube A, and the corresponding concentration of vector plasmid and corresponding P3000 (DNA:P3000 =1:2) are added to tube B, and gently pipette to mix, then add the corresponding concentration of the target plasmid, pipette to mix, and let stand for 5 min. Then add the liquid in tube A to tube B, mix gently, and let stand for 15 min. Take out the 6-well plate from the incubator, discard 500 μL of culture medium, add the liquid in tube B to the cells, and change the medium after 6 h. After 48 h of transfection, check the transfection efficiency of the cells under an optical microscope. Then collect the cell supernatant into a 1.5 mL tube, centrifuge at 1000 rpm for 5 min, and transfer the cell supernatant to a new 1.5 mL tube. The physical copy number of the virus particles in the supernatant was then quickly quantified by the qPCR method (Table 1, Table 2), which can quickly quantify the number of virus particles.

[0036] Table 1 qPCR reaction system (20 μL)

[0037] Table 2 qPCR reaction procedure .

[0038] Lentiviral transduction and screening of UPR-GFP-LNCaP stable expression cell lines; 5x10 5 / well density of LNCaP, when the cells grow to about 60%, add the lentiviral supernatant obtained in the above step and polybrene (8 μg / mL), and perform spin infection to promote lentiviral infection. After culturing overnight, replace the complete medium without polybrene for culturing, and perform puromycin antibiotic screening 48 hours after inoculation. At this time, the lowest concentration of complete lethality of 1-4 μg / mL is selected as the screening concentration, and the screened cell lines are characterized under an optical microscope to confirm the transduction efficiency. The LNCaP cell line that successfully expresses the UPR-GFP plasmid was screened for multiple passages, and the cell line with stable expression was retained for subsequent experiments.

[0039] UPR characterization: UPR-GFP-LNCaP stable expression cell line was cultured at 1x10 5 / 1ml / dish were plated on Confocal special glass dishes. After 24 hours of plating, different concentrations (5 μmol / L, 10 μmol / L) of saikosaponin D were added, and Thapsigargin (UPR inducer) was added as a positive control. Within 2-4 hours after drug addition, the Confocal glass dish containing living cells was placed under a confocal microscope for observation.

[0040] Experimental results: Live cell imaging was performed using an AX N-STORM confocal microscope (60 / 1.42NA, oil objective). A 488nm argon laser was used to excite EGFP to emit green fluorescence. Cell fluorescence images were acquired at a resolution of 1024×1024 pixels, and ImageJ software was used for image processing and analysis. Figure 4 As shown in the figure, in a single cell, compared with the group without any drug treatment, the addition of Thapsigargin can significantly activate the intracellular UPR, and the number of fluorescent spots increased significantly, which is statistically significant. After the cells were treated with saikosaponin D, the concentration of 5 μmol / L was sufficient to activate the intracellular UPR, and the number of fluorescent spots also increased significantly. The data was statistically significant. The experimental results were * for p<0.05, indicating statistical difference; ** for p<0.01, indicating significant difference; and *** for p<0.001, indicating extremely significant difference.

[0041] Figure 5 This is the interaction diagram between saikosaponin D and p-EIF2α key protein molecule.

[0042] The present invention confirms that saikosaponin D can fully activate the UPR in LNCaP cells, which may cause the failure of the endoplasmic reticulum sensor in the cells, thereby promoting cell death, and has a potential therapeutic effect on prostate cancer patients.

[0043] Example 3: Saikosaponin D can combine with the anti-clinical drug Enza to exert a better inhibitory effect on cancer cells Experimental Materials: Cell line: LNCaP (human prostate cancer cell); Culture medium: Roswell Park Memorial Institute (RPMI) 1640 + 10% FBS (Fetal Bovine Serum) containing 100 U / ml penicillin and 0.1 mg / ml streptomycin; Trypsin: 0.25% trypsin-EDTA solution; Compounds: Saikosaponin D, Enzalutamide; Incucyte® Cytotox Green Dye: used to mark dead cells in culture; Incucyte real-time live cell imaging system: used to directly measure the cell confluence or cell number changes at different time points, and provide cell proliferation curves over time; Corning® 96-well clear flat-bottom polystyrene microplate: used for real-time cell analysis experiments; CO2 incubator: used to maintain the environment required for cell growth; Microscope and cell counting chamber: used for cell counting.

[0044] LNCaP cells were inoculated into culture flasks containing RPMI 1640 medium and cultured at 37°C and 5% CO2. When the cells grew to 80%-90% confluence, they were digested with 0.25% trypsin and passaged.

[0045] Cell counting: Use 0.25% trypsin to digest the cells, wash with PBS (Phosphate Buffered Saline), and drop the cell suspension into a cell counting plate. Count the number of cells under a microscope and calculate the number of cells per milliliter.

[0046] Cell plating and proliferation assay: LNCaP cells were plated at a density of 5000 / 100 μL / well in a Corning® 96-well plate containing appropriate culture medium and placed in an Incucyte incubator overnight. Sterile DMSO was used to prepare saikosaponin D into a 20 mmol / L drug stock solution, and Enza was prepared into a 50 mmol / L drug stock solution. After 24 hours of plating, different drugs were added: 5 μmol / L saikosaponin D, 10 μmol / L Enzalutamide, 20 μmol / L Enzalutamide, 5 μmol / L saikosaponin D +10 μmol / L Enzalutamide, 5 μmol / L saikosaponin D +20 μmol / L Enzalutamide. The number of replicates in each group was n=5, and the cells were kept in a constant temperature environment during the experiment to observe the dynamic changes of the cells.

[0047] Experimental results: In the process of real-time cell monitoring, the Incucyte 10X objective lens magnification was selected, and cell proliferation was automatically detected every 4 hours for long-term continuous monitoring. The confluence percentage of the phase contrast image was used as the cell coverage (Confluence) parameter value to evaluate the changes in cell proliferation. The cell proliferation curve was plotted using the software GraphPad Prism 8.0. The experimental results were * for p < 0.05, indicating statistical difference; ** for p < 0.01, indicating significant difference; *** for p < 0.001, indicating extremely significant difference.

[0048] like Figure 6 and Figure 7 As shown, the present invention confirms that the use of saikosaponin D alone or Enzalutamide alone can inhibit the proliferation of LNCaP to a certain extent, but its inhibitory effect is not as good as the treatment group using the two drugs in combination, indicating that the combined use of saikosaponin D and Enzalutamide can synergize and exert a better inhibitory effect, and saikosaponin D as an active monomer of traditional Chinese medicine has a generally lower toxicity than chemical drugs. Enzalutamide, as an androgen receptor inhibitor, has common adverse reactions including weakness and fatigue, and may even cause male demasculinization, which is an inevitable problem for male patients. The present invention finds that if saikosaponin D is used in combination, the dose of Enzalutamide in clinical practice can be reduced, and it may have the effect of alleviating the demasculinization problem of male patients, which can bring good clinical benefits to patients.

[0049] Example 4: Example 4: Saikosaponin D combined with Enzalutamide better inhibits the growth of subcutaneous prostate cancer cell LNCaP transplanted tumors in nude mice Experimental Materials Cell line: LNCaP (human prostate cancer cell); Culture medium: Roswell Park Memorial Institute (RPMI) 1640 with 10% FBS (Fetal Bovine Serum), 100 U / ml penicillin and 0.1 mg / ml streptomycin; Buffer: Phosphate Buffer Saline (PBS); Trypsin: 0.25% trypsin-EDTA solution; Compounds: saikosaponin D, Enzalutamide; Matrigel: Corning® Matrigel® matrix.

[0050] The present invention selected 40 male nude mice (NOD / SCID mice) aged 6-8 weeks, SPF grade, and raised in the B2 animal room of South China University of Technology. The nude mice were fed with sterile granular mouse feed, ultrapure water for drinking water, and sterile corn cobs for bedding. After adaptive feeding for several days, ultrapure water and high temperature and high pressure sterilized feed were given. LNCaP cells in the logarithmic growth phase were collected, prepared into LNCaP cell suspension with PBS, and Matrigel was added at a ratio of 1:4. The cell suspension was inoculated subcutaneously in nude mice at a number of 5x10 6 cells / each. From the day when the first nude mouse tumor grew to be visible to the naked eye, the length, width and height of each nude mouse tumor were measured daily with a vernier caliper. And the tumor volume was calculated. Tumor volume = (length x width x height) x / 6. When the tumor of each mouse grew to about 300 mm, it met the inclusion criteria. The nude mice that met the inclusion criteria were randomly divided into four groups, namely the control group, the Enzalutamide group, the Bupleurum saponin D group, and the Enzalutamide and Bupleurum saponin D combination group. From the date of enrollment, each nude mouse was injected with the drug by intraperitoneal injection. Every 2 days was a dosing cycle, and a total of 15 cycles were administered. The control group was given a blank solvent on the first day of each cycle, the Enzalutamide group was given Enzalutamide 5 mg / kg on the first day of each cycle, and the Bupleurum saponin D group was given Bupleurum saponin D 5 mg / kg on the first day of each cycle. The Enzalutamide and saikosaponin D combined group was given a mixed solution of Enzalutamide 5 mg / kg + saikosaponin D 5 mg / kg on the first day of each cycle. The animals were sacrificed on the 21st day after administration, and the tumors and major organs were removed. The experimental results were expressed as "mean ± standard deviation" and statistically analyzed using SPSS 21.0 software. The variance analysis among multiple groups was compared using "one-way ANOVA".

[0051] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Application of saikosaponin D in the preparation of inducers of unfolded protein response.

2. The use of saikosaponin D according to claim 1 in the preparation of an inducer of unfolded protein response, characterized in that: The effective concentration of saikosaponin D is 1-50 μmol / L, preferably 1-20 μmol / L.

3. Application of saikosaponin D as an inducer of unfolded protein response in the preparation of drugs for the treatment of prostate cancer.

4. The use of saikosaponin D according to claim 3 as an inducer of unfolded protein response in the preparation of a drug for treating prostate cancer, characterized in that: The effective concentration of saikosaponin D is 1-50 μmol / L, preferably 1-20 μmol / L.

5. Application of saikosaponin D combined with enzalutamide in the preparation of drugs for the treatment of prostate cancer.

6. The use of saikosaponin D combined with enzalutamide in the preparation of a drug for treating prostate cancer according to claim 5, characterized in that: The effective concentration of saikosaponin D is 5-20 μmol / L; the effective concentration of enzalutamide is 10-20 μmol / L.

7. The use of saikosaponin D combined with enzalutamide in the preparation of a drug for treating prostate cancer according to claim 5, characterized in that: The effective concentration of the saikosaponin D is 5 μmol / L; the effective concentration of the enzalutamide is 20 μmol / L.

8. The use according to any one of claims 3 to 7, characterized in that: The drug for treating prostate cancer also includes pharmacodynamically acceptable auxiliary materials.

9. The use according to claim 8, characterized in that: The pharmaceutically acceptable excipient is at least one of a sustained-release agent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, an adsorption carrier, a surfactant and a lubricant.

10. The use according to any one of claims 3 to 7, characterized in that: The administration methods of the drug for treating prostate cancer include oral administration and injection administration.

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