Application of Daibaijie extract in preparing medicine for treating bone metastasis of prostate cancer

Abstract

The invention discloses an application of a Dai Baijie extract in the preparation of a drug for treating bone metastasis of prostate cancer. The invention discloses an application of a Dai Baijie extract in the preparation of a drug for treating bone metastasis of prostate cancer, and discloses that the Dai Baijie extract can effectively inhibit the proliferation, migration and invasion ability of prostate cancer PC-3 cells.

Description

Technical Field

[0001] The invention relates to application of a Daibaijie extract in preparing a medicine for treating bone metastasis of prostate cancer. Background Art

[0002] Bone metastasis is the main cause of death in prostate cancer (PCa) patients. Currently, chemotherapy is still one of the main treatments for PCa bone metastasis. However, chemotherapy can easily lead to side effects such as gastrointestinal reactions and neuropathy. Therefore, exploring effective therapeutic drugs with less toxic side effects has important clinical value. Summary of the invention

[0003] The present invention provides an application of a Daibaijie extract in the preparation of a drug for treating prostate cancer bone metastasis to solve the above-mentioned technical problems, and specifically adopts the following technical solutions:

[0004] The invention discloses an application of a Daibaijie extract in preparing a medicine for treating bone metastasis of prostate cancer.

[0005] Furthermore, the extraction method of the Daibaijie extract is:

[0006] The cut sections of Daibai were crushed, soaked in 10 times the amount of 95% ethanol, refluxed for extraction for a certain period of time, the extract was cooled and filtered, and concentrated under reduced pressure to obtain the extract concentrate, which was suspended in distilled water and extracted with an equal amount of ethyl acetate to obtain the ethyl acetate fraction.

[0007] Furthermore, the Daibaijie extract is a steroidal saponin compound.

[0008] Furthermore, the drug inhibits FDX1 protein through the Daibaijie extract to treat prostate cancer.

[0009] Furthermore, the drug inhibits FDX1 protein through the Daibaijie extract to downregulate the expression of tumor metastasis-related proteins of prostate cancer.

[0010] Furthermore, the tumor metastasis-related protein comprises at least one of Vimentin, SNAIL2, TWIST1, VEGFA, IL-11, MMP1, MMP6, MMP9, MMP10, MMP12 and MMP13.

[0011] Furthermore, the drug also includes a pharmaceutically acceptable carrier.

[0012] Furthermore, the pharmaceutically acceptable carrier includes any one or more combinations of diluents, excipients, disintegrants, fillers, binders, lubricants, flavoring agents, surfactants, and stabilizers.

[0013] Furthermore, the dosage form of the drug is any one of injection, injection, tablet, granule, pill, and capsule.

[0014] The benefit of the present invention lies in the use of the provided Dai Baijie extract in the preparation of a drug for treating bone metastasis of prostate cancer, which reveals that the Dai Baijie extract can effectively inhibit the proliferation, migration and invasion of prostate cancer PC-3 cells.

[0015] The benefit of the present invention lies in the use of the provided Dai Baijie extract in the preparation of a drug for treating prostate cancer bone metastasis, revealing that the Dai Baijie extract can inhibit the expression of FDX1 protein, thereby affecting the proliferation, migration and invasion ability of prostate cancer bone metastasis PC-3 cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Schematic diagram of the inhibition of PC-3 cell proliferation by Daibaijie extract;

[0017] Figure 2 Schematic diagram of the induction of PC-3 cell migration by Daibaijie extract;

[0018] Figure 3 Schematic diagram of the inhibition of PC-3 cell invasion by Daibaijie extract;

[0019] Figure 4 Schematic diagram showing that Daibaijie extract inhibits the expression of FDX1 in PC-3 cells;

[0020] Figure 5 Schematic diagram showing that FDX1 can reverse the inhibitory effects of Daibaijie extract on PC-3 cell invasion and migration;

[0021] Figure 6 Schematic diagram showing that FDX1 can reverse the inhibitory effect of Daibaijie extract on cytokine expression in PC-3. DETAILED DESCRIPTION

[0022] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0023] Dai Baijie is a woody vine of Tongguangsan, a plant of the genus Milkweed in the Apocynaceae family. Dai Baijie's Dai name is "Yajiexianda", which means a medicine that can cure all kinds of poisons. It is one of the most distinctive "antidote" varieties of the Dai people. It has the effects of clearing heat and detoxifying, reducing swelling and relieving pain. It is often used for sore throat, ulcers and rashes, lung heat cough, and antidote for food poisoning. This application uses prostate cancer cells PC-3 as the research object and confirms the interaction between Dai Baijie and FDX1 in PCa.

[0024] 1. Materials

[0025] 1.1 Human prostate cancer PC-3 cells (purchased from the American Type Culture Collection ATCC) were cultured in 1640 medium supplemented with 10% fetal bovine serum, 5% penicillin and streptomycin (purchased from Gibco, USA);

[0026] 1.2 RPMI 1640 culture medium, 0.25% trypsin-EDTA (purchased from Hyclone, USA);

[0027] 1.3 Fetal bovine serum, penicillin / streptomycin (purchased from Gibco, USA); FDX1 antibody (purchased from CellSignaling Technology, USA); CCK-8 cell activity detection kit (purchased from Beijing Quanshijin Biotechnology Co., Ltd., China);

[0028] 1.4 RNA extraction kit, reverse transcription kit and fluorescence quantitative PCR kit (purchased from Takar Company, Japan);

[0029] 1.5 PCR primers (purchased from Guangzhou Sihe Biotechnology Co., Ltd.);

[0030] 1.6 BCA protein concentration determination kit, ultrasensitive ECL chemiluminescence kit (purchased from Bio-Tech Biotech Co., Ltd., China);

[0031] 1.7 Cytokine antibody array (purchased from abcam);

[0032] 1.8 Dai Baijie was collected from the cultivation base of Yunnan Branch of Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences. It has a growth period of 2 years. The above-ground parts and roots of the same plant were collected, cut into sections, dried, and crushed for later use;

[0033] 1.9CO2 cell culture incubator, refrigerated high-speed centrifuge, ultra-clean biosafety workbench (Thermo Fisher, USA), fluorescence microscope (Leica, Germany), vertical gel transfer system (Biorad, USA), BioTek, USA).

[0034] 2. Experiment

[0035] 2.1 Preparation, extraction and identification of Daibaijie extract

[0036] The cut sections of Daibai were crushed, 400g of powder was taken, 10 times the amount of 95% ethanol was added for infiltration, reflux extraction was performed for 2h, the extract was cooled and filtered, and the extract was concentrated under reduced pressure to obtain an extract, and the extraction rate was 13.0%. After the extract was suspended with 500mL of distilled water, it was extracted with an equal amount of ethyl acetate to obtain the ethyl acetate fraction (EFA), that is, the yield of the present application was 5.9%. The types of chemical components that may exist in the active site of EFA were detected by test tube method, filter paper method and other methods, and the results showed that EFA mainly contained steroidal saponins and organic acid compounds.

[0037] 2.2CCK8 detection of cell viability

[0038] Use 0.25% trypsin to digest PC-3 cells to prepare a cell suspension, inoculate them into a 96-well culture plate at a density of 3×103 cells / well, and add drugs according to the following groups after the cells adhere to the wall. Normal control group: culture cells are not treated with drugs, only culture fluid is added. Drug administration group: divided into 6 dose groups, add Dai Baijie extract EAF to the culture fluid, and the final concentration of the drug solution is 0.75, 1.5, 3, 6, 12, 24μg / mL, respectively. After continuing to culture for 24h, 10μL CCK8 reagent is added to each well and incubated for 2 hours, and then the absorbance value at a wavelength of 450nm is detected with an enzyme reader, and 3 replicates are set for each group.

[0039] 2.3 Transwell assay

[0040] After digesting PC-3 cells with 0.25% trypsin, a cell suspension was prepared. 100 μL of the adjusted PC-3 cell suspension of 1×106 cells / mL was inoculated in the upper chamber of the Transwell. Different concentrations of EAF solution of Daibaijie extract (0, 1.5, 3, 6 μg / mL) were added to the lower chamber, and then cultured for 24 hours. The Transwell chamber was taken out, the PC-3 cells were fixed for 30 minutes, and the PC-3 cells on the upper chamber surface were carefully removed after washing. The specimens were stained with 0.1% crystal violet stain for 20 minutes, dried, and the cell characteristics were observed under an inverted microscope. Then 5 high-power fields were randomly selected, and the PC-3 cells were photographed and the number was calculated to calculate the average value. When detecting the migration ability of cells, it is not necessary to add Matrigel matrix gel in the upper chamber, but when detecting the invasion ability of cells, Matrigel matrix gel dilution (1:5) needs to be coated in the upper chamber of the Transwell, and the other conditions are the same as the migration test.

[0041] 2.4qRT-PCR analysis

[0042] PC-3 cells were co-cultured with EAF solutions of 0, 1.5, 3, and 6 μg / mL of Daibaijie extract for 24 h, and total RNA was extracted from the cells under low temperature, and then the total RNA samples were immediately reverse transcribed into cDNA. PCR amplification was performed using a real-time PCR kit for a total of 40 cycles. The relative expression level of FDX1 mRNA was calculated using the 2-ΔΔCT method.

[0043] 2.5 Cell transfection

[0044] PC-3 cells were digested with 0.25% trypsin to prepare a cell suspension, which was inoculated into a 6-well plate (1×106 cells / well). The cell density was cultured to 40% in a 37°C, 5% CO2 incubator. FDX1 and FDX1-RNAi were transfected into PC-3 cells using Lipofectamine 3000 transfection reagent.

[0045] 2.6 Western blot analysis

[0046] At 100mm 2 Four groups of PC-3 cells were inoculated in the culture dishes, and the total protein of PC-3 cells in each group after 24 hours of treatment was extracted using mixed lysate, and the total protein was quantified by BCA standard method. It was electrophoresed in 10% to 12% SDS-PAGE gel, then electrotransferred to PVDF membrane, blocked with 2% BSA after 2 hours, and added with primary antibody diluent FDX1 after 1 hour, and incubated in low temperature and away from light. Then the secondary antibody was added and shaken at low speed for 1 hour, washed 3 times, and ECL luminescence detection was selected to expose the band imaging. All experiments were repeated 3 times.

[0047] 2.7 Cytokine detection

[0048] First, after treating the cells for 24 hours according to the following EAF group, si-FDX1 group, FDX1 group, si-FDX1+EAF group and FDX1+EAF group, the cell supernatant of each treatment group was collected and the expression of 11 cytokines was detected by cytokine antibody array. The operation method was carried out according to the standard protocol of the reagents.

[0049] 2.8 Statistical methods

[0050] SPSS 20.0 and GraphPad Prism were used for statistical analysis and drawing, and the data were expressed as mean ± standard deviation. The data were analyzed by t-test and one-way analysis of variance, and P < 0.05 indicated that the difference was statistically significant.

[0051] 3. Results

[0052] 3.1 EAF from Daibaijie extract inhibits PC-3 cell proliferation

[0053] PC-3 cells were treated with 0, 0.75, 1.5, 3, 6, and 12 μg / mL EAF solution, and then cell proliferation was detected by CCK8 assay. Figure 1 As shown in the figure, compared with the control group, EAF had an inhibitory effect on the proliferation of PC-3 cells in a concentration-dependent manner (P<0.01).

[0054] 3.2 EAF, an extract of Daibaijie, inhibits PC-3 cell migration

[0055] After PC-3 cells were treated with 0, 1.5, 3, and 6 μg / mL EAF solution for 24 h, the Figure 2 As shown in the figure, the results of Transwell experiment showed that EAF could inhibit the migration ability of PC-3 cells, and the difference was statistically significant compared with the control group (P<0.01).

[0056] 3.3 EAF from Daibaijie extract inhibits PC-3 cell invasion

[0057] After PC-3 cells were treated with 0, 1.5, 3, and 6 μg / mL EAF solution for 24 h, the invasion ability of PC-3 cells was detected using matrix-coated Transwell. Figure 3 As shown in the figure, EAF could inhibit the invasion ability of PC-3 cells, and the difference was statistically significant compared with the control group (P<0.01).

[0058] 3.4 EAF from Daibaijie inhibits the expression of FDX1, a key gene for copper-induced death in PC-3 cells

[0059] PC-3 cells were treated with EAF solutions of different concentrations (0, 1.5, 3, and 6 μg / mL) for 24 h, and then the expression of gene FDX1 in PC-3 cells was detected by PCR and Western blot. Figure 4 As shown in the figure, compared with the control group, EAF inhibited the expression of FDX1 mRNA in a concentration-dependent manner (P<0.01). At the same time, EAF downregulated the expression of FDX1 protein in PC-3 cells in a concentration-dependent manner, and the difference was statistically significant compared with the control group.

[0060] 3.5 Overexpression of FDX1 reverses the inhibitory effect of EAF extracted from Daibaijie on PC-3 cell migration and invasion

[0061] EAF group, si-FDX1 group, FDX1 group, si-FDX1+EAF group and FDX1+EAF group were set up, and the effects of each group on the invasion and migration of PC-3 cells were studied by Transwell assay. Figure 5As shown in the data, compared with the control group, the invasion and migration abilities of PC-3 cells in the EAF group, si-FDX1 group and si-FDX1+EAF group were significantly inhibited (P < 0.01), while the invasion and migration abilities of cells in the FDX1 group and FDX1+EAF group were not significantly different from those in the control group (P > 0.05).

[0062] 3.6 EAF extract from Daibaijie inhibits the expression of cytokines in PC-3 cells

[0063] In order to further study the effects of EAF and FDX1 on the expression of cytokines in PC-3 cells, the present application tested the effects of EAF on the expression of Vimentin, SNAIL2, TWIST1, VEGFA, IL-11, MMP1, MMP6, MMP9, MMP10, MMP12 and MMP13 in PC-3 cells. Figure 6 As shown in the figure, the expression of the above cytokines in PC-3 cells in the EAF group, si-FDX1 group and si-FDX1+EAF group was significantly inhibited, while the expression of cytokines in the FDX1 overexpression group was significantly increased, among which TWIST1 and MMP10 were the most significant.

[0064] In the experiment of the present application, an EAF solution was prepared, and then the effect of 0, 0.75, 1.5, 3, 6, and 12 μg / mL EAF solution on the proliferation of PC-3 cells was detected by CCK8. The results showed that EAF could significantly inhibit the proliferation of PC-3 cells. On this basis, PC-3 cells were treated with 0, 1.5, 3, and 6 μg / mL EAF solution, and the effect of EA on the metastatic ability of PC-3 cells was detected by Transwell experiment. The results showed that EAF could inhibit the migration and invasion ability of PC-3 cells. The above results show that the EAF solution derived from Dai Baijie has a certain anti-prostate cancer effect.

[0065] In the experiment of this application, the expression of FDX1 in PC-3 cells treated with 0, 1.5, 3, and 6 μg / mL EAF solution was detected by PCR and Western blot. The results showed that EAF inhibited the expression of FDX1 mRNA and protein in a concentration-dependent manner. Therefore, the anti-tumor effect of EAF on PC-3 cells is related to the inhibition of the expression of FDX1, a key regulator of copper death, that is, EAF may affect the proliferation, migration and invasion of prostate cancer bone metastasis PC-3 cells by inhibiting the expression of FDX1 protein.

[0066] A variety of cell proteins such as Vimentin, SNAIL2, TWIST1, VEGFA, IL-11 and MMPs are closely related to the malignant behavior of tumors. They are involved in regulating tumor cell growth, proliferation, invasion, angiogenesis and metastasis. MMPs are a class of zinc-dependent endogenous proteases that play multiple roles in tissue remodeling and the degradation of various proteins in the extracellular matrix. It is currently known that MMPs can promote cell proliferation, migration and differentiation, and play a role in apoptosis, angiogenesis, tissue repair and immune response. At the same time, they affect the bioactive molecules on the cell surface and regulate various cells and signal transduction pathways. MMP-1, MMP-6, MMP-9, MMP-10, MMP-11 and MMP-12 are members of the MMPs family. MMPs can hydrolyze extracellular matrix-related components to enable tumor cells to have a stronger ability to invade surrounding tissues. In addition, Twist1 is a member of the basic helix-loop-helix transcription factor family and is an important transcription factor that induces EMT, migration and invasion of cancer cells. Twist1 is highly expressed in many invasive cancers and acts as an oncogene. In the experiment of this application, the expression of 11 cytokines was detected by cytokine antibody array. The results showed that EAF and inhibition of FDX1 can downregulate the expression of tumor metastasis-related proteins, while the expression of tumor metastasis-related proteins is significantly upregulated after upregulating FDX1. At the same time, EAF and upregulating FDX1 have the effect of synergistically inhibiting the expression of tumor metastasis-related proteins. Therefore, EAF can affect the proliferation, migration and invasion adhesion ability of prostate cancer bone metastasis PC-3 cells by inhibiting the expression of tumor metastasis-related proteins.

[0067] In summary, EAF, an extract of Daibaijie, can inhibit the proliferation, migration and invasion of PC-3 cells. Its mechanism of action is related to EAF's inhibition of the expression of FDX1 and tumor metastasis-related proteins, which provides a theoretical basis and new hope for the clinical treatment of prostate cancer with EAF.

[0068] The present application proposes an application of a Dai Baijie extract in the preparation of a drug for treating bone metastasis of prostate cancer. The extraction method of the Dai Baijie extract is: the Dai Baijie is cut into pieces and crushed, 10 times the amount of 95% ethanol is added for infiltration, reflux extraction is performed for a certain period of time, the extract is cooled and filtered, and the extract is concentrated under reduced pressure to obtain an extract, the extract is suspended in distilled water, and then extracted with an equal amount of ethyl acetate to obtain an ethyl acetate fraction. Furthermore, the Dai Baijie extract is a steroidal saponin compound.

[0069] The drug inhibits FDX1 protein through Daibaijie extract to treat prostate cancer bone metastasis.

[0070] Furthermore, the drug inhibits FDX1 protein through the Daibaijie extract to downregulate the expression of tumor metastasis-related proteins of prostate cancer. The tumor metastasis-related proteins include at least one of Vimentin, SNAIL2, TWIST1, VEGFA, IL-11, MMP1, MMP6, MMP9, MMP10, MMP12 and MMP13.

[0071] The drug also includes a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier includes any one or more combinations of diluents, excipients, disintegrants, fillers, adhesives, lubricants, flavoring agents, surfactants, and stabilizers. The dosage form of the drug is any one of injection, injection, tablet, granule, granule, pill, and capsule.

[0072] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any form, and any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

Claims

1. Use of a Daibaijie extract in the preparation of a drug for treating bone metastasis of prostate cancer; The drug inhibits FDX1 protein through the Daibaijie extract to treat prostate cancer bone metastasis; The drug inhibits FDX1 protein through the Daibaijie extract to downregulate the expression of tumor metastasis-related proteins of prostate cancer; The tumor metastasis-related protein comprises at least one of Vimentin, SNAIL2, TWIST1, VEGFA, IL-11, MMP1, MMP6, MMP9, MMP10, MMP12 and MMP13; The extraction method of the Daibaijie extract is: The cut segments of Daibai were crushed, soaked in 10 times the amount of 95% ethanol, refluxed for a certain period of time, the extract was cooled and filtered, and concentrated under reduced pressure to obtain the extract concentrate. The extract concentrate was suspended in distilled water and extracted with an equal amount of ethyl acetate to obtain the ethyl acetate fraction. The Daibaijie extract is a steroidal saponin compound.

2. The use of the Daibaijie extract according to claim 1 in the preparation of a medicament for treating bone metastasis of prostate cancer, characterized in that: The medicament further includes a pharmaceutically acceptable carrier.

3. The use of the Daibaijie extract according to claim 2 in the preparation of a medicament for treating bone metastasis of prostate cancer, characterized in that: The pharmaceutically acceptable carrier includes any one or more combinations of diluents, excipients, disintegrants, fillers, binders, lubricants, flavoring agents, surfactants, and stabilizers.

4. The use of the Daibaijie extract according to claim 2 in the preparation of a medicament for treating bone metastasis of prostate cancer, characterized in that: The dosage form of the medicine is any one of tablets, granules, pills and capsules.

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