CN116421640B - Application of Daibaijie extract in preparing medicine for treating bone metastasis of prostate cancer - Google Patents

Abstract

The invention discloses application of Dai Baijie extract in preparing a medicament for treating prostate cancer bone metastasis. The invention discloses application of Dai Baijie extract in preparing a medicament for treating prostate cancer bone metastasis, and discloses the Dai Baijie extract which can effectively inhibit proliferation, migration and invasion capacity of prostate cancer PC-3 cells.

Description

Application of Dai Baijie extract in preparation of medicine for treating prostate cancer bone metastasis

Technical Field

The invention relates to application of Dai Baijie extract in preparing a medicament for treating prostate cancer bone metastasis.

Background

Bone metastasis is the major cause of death in Prostate cancer (PCa) patients, and chemotherapy is still one of the main treatments for PCa bone metastasis. However, chemotherapy is easy to cause side effects such as gastrointestinal reaction, neuropathy and the like, so that the research of effective therapeutic drugs with small toxic and side effects has important clinical value.

Disclosure of Invention

The invention provides an application of Dai Baijie extract in preparing a medicament for treating prostate cancer bone metastasis, which solves the technical problems, and specifically adopts the following technical scheme:

application of Dai Baijie extract in preparing medicine for treating prostate cancer bone metastasis is provided.

Further, the extraction method of the Dai Baijie extract comprises the following steps:

Cutting Dai hundred, pulverizing, adding 10 times of 95% ethanol, reflux extracting for a certain time, cooling the extractive solution, filtering, concentrating under reduced pressure to obtain extract, suspending the extract with distilled water, and extracting with equal amount of ethyl acetate to obtain ethyl acetate part.

Further, the Dai Baijie extract is a steroid saponin compound.

Further, the medicine inhibits FDX1 protein by the Dai Baijie extract to treat prostatic cancer.

Further, the medicine inhibits the FDX1 protein through the Dai Baijie extract to down regulate the expression of tumor metastasis related protein of the prostate cancer.

Further, the tumor metastasis associated protein comprises at least one of Vimentin, SNAIL, TWIST1, VEGFA, IL-11, MMP1, MMP6, MMP9, MMP10, MMP12, and MMP 13.

Further, the medicament also comprises a pharmaceutically acceptable carrier.

Further, the pharmaceutically acceptable carrier comprises any one or more combinations of diluents, excipients, disintegrants, fillers, binders, lubricants, flavoring agents, surfactants, stabilizers.

Further, the medicament is any one of injection, tablet, granule, pill and capsule.

The invention has the advantages that the application of the Dai Baijie extract in preparing the medicine for treating the prostate cancer bone metastasis reveals that the Dai Baijie extract can effectively inhibit the proliferation, migration and invasion capacity of the prostate cancer PC-3 cells.

The invention has the advantages that the application of the Dai Baijie extract in preparing the medicine for treating the prostate cancer bone metastasis reveals that the Dai Baijie extract can inhibit the FDX1 protein expression, thereby affecting the proliferation, migration and invasion capacity of the prostate cancer bone metastasis PC-3 cells.

Drawings

FIG. 1 is a schematic representation of the inhibition of PC-3 cell proliferation by Dai Baijie extract;

FIG. 2 is a schematic representation of the induction of PC-3 cell migration by Dai Baijie extract;

FIG. 3 is a schematic representation of the inhibition of PC-3 cell invasion by Dai Baijie extract;

FIG. 4 is a schematic representation of Dai Baijie extract inhibiting the expression of FDX1 in PC-3 cells;

FIG. 5 is a schematic representation of the inhibition of PC-3 cell invasion and migration by FDX1 reversible Dai Baijie extract;

FIG. 6 is a schematic representation of the inhibition of cytokine expression in PC-3 by FDX1 reversible Dai Baijie extract.

Detailed Description

The invention is described in detail below with reference to the drawings and the specific embodiments.

Dai Baijia is woody vine of light-transmitting powder of plant of genus Dairy of family Asclepiadaceae, dai Baijia Dai name is "elegance first beat", which means drug for detoxifying, is one of the most characteristic drug-detoxifying varieties of Dai nationality, has the effects of clearing fire, detoxicating, detumescence and relieving pain, and is commonly used for treating sore throat, treating sore rash, cough due to lung heat, detoxifying drug and food toxin and the like. The application takes the prostate cancer cell PC-3 as a research object, and confirms the interaction of Dai Baijie and FDX1 in PCa.

1. Material

1.1 Human prostate cancer PC-3 cells (purchased from American type culture Collection) were cultured using 1640 medium (purchased from Gibco corporation, USA) supplemented with 10% fetal bovine serum, 5% penicillin and streptomycin;

1.2RPMI 1640 medium, 0.25% trypsin-EDTA (available from Hyclone Inc. of America);

1.3 fetal bovine serum, penicillin/streptomycin (from Gibco, U.S.), FDX1 antibody (from CELL SIGNALING Technology, U.S.), CCK-8 cell activity assay kit (from Beijing full gold Biotechnology Co., ltd.);

1.4RNA extraction kit, reverse transcription kit and fluorescent quantitative PCR kit (purchased from Takar Co., japan);

1.5PCR primers (purchased from Guangzhou, inc. of Biotech Co., ltd.);

1.6BCA protein concentration assay kit, hypersensitive ECL chemiluminescent kit (purchased from Biyun Tian Biotech Co., china);

1.7 cytokine antibody arrays (available from abcam corporation);

1.8 Dai Baijie is taken from a Yunnan part cultivation base of medical plant institute of China academy of medical science, the growth period is 2 years, the overground parts and roots of the same plant are collected, cut into sections, dried in the sun and crushed for later use;

1.9CO2 cell incubator, refrigerated high speed centrifuge, ultra clean biosafety table (Thermo Fisher, usa), fluorescence microscope (Leica, germany), vertical gel transfer system (Biorad, usa), bioTek, usa).

2. Experiment

2.1 Preparation, extraction and identification of Dai Baijie extract

Cutting Dai Baijiegu into segments, crushing, taking 400g of powder, adding 10 times of 95% ethanol for soaking, reflux-extracting for 2h, cooling the extracting solution, filtering, and concentrating under reduced pressure to obtain an extract with the extraction rate of 13.0%. The extract is suspended by 500mL of distilled water, and is extracted by using equal amount of ethyl acetate to obtain an ethyl acetate part (ETHYL ACETATE Fraction, EFA), namely the yield of the application is 5.9%. The detection of possible chemical component types in EFA active site by test tube method, filter paper sheet method, etc. shows that EFA contains mainly steroid saponin and organic acid compound.

2.2CCK8 detection of cell viability

Cell suspensions were prepared using 0.25% pancreatin to digest PC-3 cells, seeded into 96-well culture plates at a density of 3 x 103 cells/well, and dosed as follows after cell attachment. Normal control group, cultured cells were not treated with drug, only with culture solution. The administration group comprises 6 dose groups, dai Baijie extract EAF is added into the culture solution, the final concentration of the liquid medicine is respectively 0.75, 1.5, 3, 6, 12 and 24 mug/mL, 10 mug of CCK8 reagent is added into each hole after continuous culture for 24 hours for incubation for 2 hours, and then an enzyme-labeled instrument is used for detecting the absorbance value at the wavelength of 450nm, and 3 replicates are arranged in each group.

2.3Transwell test

PC-3 cells were digested with 0.25% pancreatin and then prepared into a cell suspension, 100. Mu.L of the prepared PC-3 cell suspension having a density of 1X 106 cells/mL was inoculated into the upper chamber of a Transwell, and the EAF solutions (0, 1.5, 3, 6. Mu.g/mL) of Dai Baijie extract having different concentrations were added to the lower chamber, followed by further culturing for 24 hours. Taking out a Transwell cell, fixing PC-3 cells for 30min, carefully removing PC-3 cells on the upper surface of the cell after washing, selecting 0.1% crystal violet stain to dye a specimen for 20min, airing, observing the characteristics of the cells under an inverted microscope, randomly selecting 5 high-power visual fields, photographing the PC-3 cells, calculating the number, and calculating the average value. In testing the migration capacity of cells, matrigel matrix gel is not required to be added into an upper chamber, while in testing the invasion capacity of cells, matrigel matrix gel diluent (1:5) is required to be coated into a Transwell upper chamber, and other conditions are the same as migration test.

2.4QRT-PCR analysis

PC-3 cells are respectively cultured for 24 hours with Dai Baijie extract EAF solution with concentration of 0, 1.5, 3 and 6 mug/mL, total RNA in the cells is extracted under low temperature environment, and then the total RNA sample is 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.

2.5 Cell transfection

PC-3 cells were digested with 0.25% pancreatin to prepare a cell suspension, inoculated into 6-well plates (1X 106 cells/well), cultured in a 5% CO2 incubator at 37℃to a cell density of 40%, and FDX1, FDX1-RNAi was transfected into PC-3 cells using Lipofectamine 3000 transfection reagent.

2.6Western blot analysis

Four groups of PC-3 cells were inoculated into 100mm ² dishes, and total protein of PC-3 cells after 24 hours of treatment was extracted from each group using a mixed lysate, and quantified by BCA standard method. Electrophoresis is carried out on the PVDF membrane in 10% -12% SDS-PAGE gel, then the PVDF membrane is subjected to electrotransformation, 2% BSA is used for blocking after 2 hours, primary anti-diluent FDX1 is added after 1 hour, and the PVDF membrane is incubated in a dark low temperature. Then adding a secondary antibody, vibrating for 1h at a low speed, washing for 3 times, and selecting ECL (electron cyclotron resonance) luminescence detection for imaging an exposure strip. All experiments were repeated 3 times.

2.7 Cytokine detection

After first treating cells for 24 hours in the following groups of EAF, si-FDX1, si-FDX1+EAF and FDX1+EAF, the cell supernatants of each treatment group were collected and the expression of 11 cytokines was detected by cytokine antibody arrays, and the procedure was performed according to the reagent standard protocol.

2.8 Statistical methods

Statistical analysis and mapping were performed using SPSS 20.0 and GRAPHPAD PRISM, and data for the data were expressed as mean.+ -. Standard deviation. The data were analyzed by t-test, single factor analysis of variance method, with P <0.05 representing the difference statistically significant.

3. Results

3.1 Dai Baijie extract EAF inhibiting PC-3 cell proliferation

PC-3 cells were treated with 0, 0.75, 1.5, 3,6, 12. Mu.g/mL EAF solution, respectively, and then examined for cell proliferation by CCK8 assay. As shown in FIG. 1, EAF has an inhibitory effect on PC-3 cell proliferation and shows a concentration dependence (P < 0.01) as compared to the control group.

3.2 Dai Baijie extract EAF inhibiting PC-3 cell migration ability

After 24h treatment of PC-3 cells with 0, 1.5, 3, 6. Mu.g/mL EAF solution, respectively, as shown in FIG. 2, the results of the Transwell experiments showed that EAF inhibited the migration capacity of PC-3 cells, and that the differences were statistically significant (P < 0.01) compared to the control group.

3.3 Dai Baijie extract EAF ability to inhibit PC-3 cell invasion

PC-3 cells were treated with 0, 1.5, 3, 6. Mu.g/mL EAF solution for 24h, respectively, and then tested for invasive capacity using matrix-coated Transwell. As shown in FIG. 3, EAF inhibited the invasive capacity of PC-3 cells, the difference was statistically significant (P < 0.01) compared to the control group.

3.4 Dai Baijie extract EAF inhibiting the expression of copper death critical gene FDX1 in PC-3 cells

PC-3 cells were treated with EAF solutions at various concentrations of 0, 1.5, 3, 6. Mu.g/mL for 24h, and then tested for expression of gene FDX1 in PC-3 cells by PCR and Western blot experiments. As shown in fig. 4, EAF inhibited FDX1 mRNA expression in a concentration-dependent manner (P < 0.01) compared to the control group. Meanwhile, EAF is concentration-dependent to down-regulate the expression of FDX1 protein in PC-3 cells, and the difference is statistically significant compared with a control group.

3.5 Over-expression of FDX1 to reverse the inhibition of PC-3 cell migration and invasion by Dai Baijie extract EAF

The EAF group, the si-FDX1 group, the si-FDX1+EAF group and the FDX1+EAF group were set, and the effect of each group on invasion and migration of PC-3 cells was studied by a Transwell experiment. As shown in FIG. 5, the invasion and migration ability of PC-3 cells in the EAF group, si-FDX1 group and si-FDX1+EAF group were significantly inhibited (P < 0.01) compared to the control group, while the invasion and migration ability of cells in the FDX1 group and FDX1+EAF group were not statistically different (P > 0.05) compared to the control group.

3.6 Dai Baijie extract EAF inhibiting expression of PC-3 cytokine

To further investigate the effect of EAF and FDX1 on the intracellular cytokine expression in PC-3, the present application experimentally examined the effect of EAF on changes in expression of Vimentin, SNAIL2, TWIST1, VEGFA, IL-11, MMP1, MMP6, MMP9, MMP10, MMP12 and MMP13 in PC-3 cells. As shown in FIG. 6, the results showed that the expression of the above cytokines was significantly inhibited in PC-3 cells in EAF, si-FDX1 and si-FDX1+EAF groups, while the expression of the cytokines in the over-expressed FDX1 groups was significantly increased. Of these, TWIST1 and MMP10 are the most remarkable.

In the experiments of the present application, EAF solutions were prepared and then the effect of 0, 0.75, 1.5, 3, 6, 12. Mu.g/mL EAF solution on PC-3 cell proliferation was examined by CCK8, with the result that EAF was able to significantly inhibit PC-3 cell proliferation. Based on this, PC-3 cells were treated with 0, 1.5, 3, 6. Mu.g/mL of EAF solution, respectively, and the effect of EA on the transfer capacity of PC-3 cells was examined by a Transwell experiment, which showed that EAF inhibited the migration and invasion capacity of PC-3 cells. The results show that the EAF solution from Dai Baijie has a certain effect of resisting prostate cancer.

In the test of the present application, the expression of FDX1 was examined by PCR and Western blot with 0, 1.5, 3, 6. Mu.g/mL of EAF solution treated with PC-3 fine, and as a result, it was found that EAF inhibited the expression of FDX1 mRNA and protein in a concentration-dependent manner. Thus, the anti-neoplastic effect exerted by EAF on PC-3 cells is associated with the inhibition of the expression of FDX1, a key regulator of copper death, i.e., EAF may affect the proliferation, migration and invasiveness of prostate cancer bone metastasis PC-3 cells by inhibiting FDX1 protein expression.

Various cellular proteins such as Vimentin, SNAIL, TWIST1, VEGFA, IL-11, and MMPs are closely related to malignant behaviors of tumors, and they are involved in regulating tumor cell growth, proliferation, invasion, angiogenesis, and metastasis. MMPs are a class of zinc-dependent endogenous proteases that have multiple roles in tissue remodeling and degradation of various proteins of the extracellular matrix. MMPs are known to promote cell proliferation, migration and differentiation and to play a role in apoptosis, angiogenesis, tissue repair and immune responses, while affecting bioactive molecules on the cell surface and modulating various cell and signaling pathways. MMP-1, MMP-6, MMP-9, MMP-10, MMP-11 and MMP-12 are members of the MMPs family, and MMP can hydrolyze extracellular matrix related components so that tumor cells have stronger invasion capacity to surrounding tissues. In addition, twist1 is a member of the basic helix-loop-helix transcription factor family, an important transcription factor that induces cancer cell EMT, migration, and invasion, twist1 is highly expressed in many invasive cancers and acts as an oncogene. In the experiment of the application, the expression of 11 cytokines is detected through a cytokine antibody array, and the result shows that EAF and FDX1 inhibition can down regulate the expression of tumor metastasis related proteins, the expression of tumor metastasis related proteins is obviously up-regulated after FDX1 is up-regulated, and the EAF and FDX1 up-regulated have the function of synergistically inhibiting the expression of tumor metastasis related proteins. Thus, EAF can affect the proliferation, migration and invasive adhesion capacity of prostate cancer bone metastasis PC-3 cells by inhibiting tumor metastasis associated protein expression.

In conclusion, the Dai Baijie extract EAF can inhibit proliferation, migration and invasion of PC-3 cells, and the action mechanism of the Dai Baijie extract EAF is related to the inhibition of FDX1 and tumor metastasis related protein expression by EAF, so that theoretical basis and new hope are provided for the clinical treatment of prostate cancer by EAF.

The application provides application of Dai Baijie extract in preparing a medicament for treating prostate cancer bone metastasis. The extraction method of the Dai Baijie extract comprises cutting Dai Baijie into segments, pulverizing, adding 10 times of 95% ethanol for soaking, reflux extracting for a certain time, cooling the extractive solution, filtering, concentrating under reduced pressure to obtain extract, suspending the extract with distilled water, and extracting with equal amount of ethyl acetate to obtain ethyl acetate part. Further, dai Baijie extract is a steroid saponin compound.

The medicine can inhibit FDX1 protein by Dai Baijie extract to treat prostate cancer bone metastasis.

Further, the medicine inhibits FDX1 protein through Dai Baijie extract to down regulate the expression of tumor metastasis related protein of prostate cancer. Tumor metastasis associated proteins comprise at least one of Vimentin, SNAIL, TWIST1, VEGFA, IL-11, MMP1, MMP6, MMP9, MMP10, MMP12, and MMP 13.

The medicament also comprises a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include any one or more combinations of diluents, excipients, disintegrants, fillers, binders, lubricants, flavoring agents, surfactants, stabilizers. The medicine is in the form of injection, tablet, granule, pill, or capsule.

The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be appreciated by persons skilled in the art that the above embodiments are not intended to limit the invention in any way, and that all technical solutions obtained by means of equivalent substitutions or equivalent transformations fall within the scope of the invention.

Claims (4)

1. Application of Dai Baijie extract in preparing medicament for treating prostate cancer bone metastasis;

The medicine inhibits FDX1 protein through the Dai Baijie extract to treat prostate cancer bone metastasis;

the medicine inhibits FDX1 protein through the Dai Baijie extract to down regulate the expression of tumor metastasis related protein of prostate cancer;

The tumor metastasis associated protein comprises at least one of Vimentin, SNAIL2, TWIST1, VEGFA, IL-11, MMP1, MMP6, MMP9, MMP10, MMP12, and MMP 13;

The extraction method of the Dai Baijie extract comprises the following steps:

Cutting Dai hundred, pulverizing, adding 10 times of 95% ethanol, reflux extracting for a certain time, cooling the extractive solution, filtering, concentrating under reduced pressure to obtain extract, suspending the extract with distilled water, and extracting with equal amount of ethyl acetate to obtain ethyl acetate part;

the Dai Baijie extract is a steroid saponin compound.

2. The use of Dai Baijie extract according to claim 1 in the preparation of a medicament for the treatment of prostate cancer bone metastasis, it is characterized in that the method comprises the steps of,

The medicament also comprises a pharmaceutically acceptable carrier.

3. The use of Dai Baijie extract according to claim 2 in the preparation of a medicament for the treatment of prostate cancer bone metastasis, it is characterized in that the method comprises the steps of,

The pharmaceutically acceptable carrier comprises any one or more of a diluent, an excipient, a disintegrant, a filler, a binder, a lubricant, a flavoring agent, a surfactant and a stabilizer.

4. The use of Dai Baijie extract according to claim 2 in the preparation of a medicament for the treatment of prostate cancer bone metastasis, it is characterized in that the method comprises the steps of,

The dosage form of the medicine is any one of tablets, granules, pills and capsules.