CN116036070A - Application of 9' -salvianolic acid B monomethyl ester in preparation of medicines for treating breast cancer tumor cell proliferation, migration and invasion - Google Patents
Application of 9' -salvianolic acid B monomethyl ester in preparation of medicines for treating breast cancer tumor cell proliferation, migration and invasion Download PDFInfo
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- 230000009401 metastasis Effects 0.000 claims abstract description 4
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- 239000000546 pharmaceutical excipient Substances 0.000 claims description 2
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
- A61K31/343—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide condensed with a carbocyclic ring, e.g. coumaran, bufuralol, befunolol, clobenfurol, amiodarone
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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Abstract
The invention discloses an application of 9' -salvianolic acid B monomethyl in preparing medicines for treating breast cancer tumor cell proliferation, migration and invasion. The invention discovers that 9 '-salvianolic acid B monomethyl ester targets Daam1 for the first time and inhibits proliferation, migration and invasion of breast cancer tumor cells, which has important significance for treating breast cancer and improving prognosis of patients, so that 9' -salvianolic acid B monomethyl ester can be used for preparing medicaments for treating breast cancer or inhibiting breast cancer metastasis.
Description
Technical Field
The invention belongs to the field of medicaments, and relates to application of 9' -salvianolic acid B monomethyl ester in preparation of medicaments for treating breast cancer tumor cell proliferation, migration and invasion.
Background
Recent data from the 2018 international cancer research Institute (IARC) survey showed that breast cancer had a 24.2% incidence of global female cancers, with 52.9% occurring in developing countries. In China, the incidence rate of breast cancer is in an increasing trend year by year, and more than 30 ten thousand women are diagnosed with the breast cancer each year. The incidence of breast cancer is particularly pronounced in eastern coastal areas and large, economically developed cities. From the aspect of the age of onset, the incidence rate of breast cancer in China gradually rises from 20 years old and reaches a high value from 45 to 50 years old. With the popularity of new therapeutic strategies and approaches, the mortality rate of breast cancer has gradually decreased worldwide. However, in china, and particularly in vast rural areas, the mortality decline trend for breast cancer is not significant. The 9' -salvianolic acid B monomethyl ester is an active ingredient in ethanol extract of Saviae Miltiorrhizae radix (Radix Salviae Miltiorrhizae). The effect of 9' -salvianolic acid B monomethyl ester on breast cancer tumor cells has not been reported in the prior art.
Disclosure of Invention
The invention aims at overcoming the defects in the prior art and provides application of 9' -salvianolic acid B monomethyl ester in preparing medicaments for treating breast cancer tumor cell proliferation, migration and invasion.
The aim of the invention can be achieved by the following technical scheme:
application of 9' -salvianolic acid B monomethyl ester in preparing medicine for treating breast cancer.
Application of 9' -salvianolic acid B monomethyl ester in preparing medicines for inhibiting breast cancer metastasis and/or invasion.
Application of 9' -salvianolic acid B monomethyl ester in preparing medicines for inhibiting breast cancer proliferation is provided.
A pharmaceutical composition comprises 9 "-monomethyl salvianolic acid B.
As a preferable mode, the pharmaceutical composition also contains pharmaceutical excipients required by preparing 9' -salvianolic acid B monomethyl ester into pharmaceutical preparations.
The beneficial effects are that:
the invention discovers that 9 '-salvianolic acid B monomethyl ester targets Daam1 for the first time and inhibits proliferation, migration and invasion of breast cancer tumor cells, which has important significance for treating breast cancer and improving prognosis of patients, so that 9' -salvianolic acid B monomethyl ester can be used for preparing medicines for treating breast cancer or inhibiting proliferation and metastasis of breast cancer.
Drawings
FIG. 1 shows the chemical structural formulae (A) and 9 "-salvianolic acid B methyl ester of Daam 1.
FIG. 2.MST experiment results
Panel A shows a graph of the binding of the full length of Daam1 to 9 "-salvianolic acid B methyl ester, and panel B shows a graph of the binding of the Daam1-FH2 domain to 9" -salvianolic acid B methyl ester.
FIG. 3.9 "-Monomethyl salvianolic acid B inhibiting proliferation, migration and invasion of breast cancer MDA-MB-231 cells.
(A, B) MDA-MB-231 breast cancer cells are respectively induced by 9' -salvianolic acid B monomethyl ester with different concentrations for 24, 48 and 72 hours, and then the inhibition rate of the drug to the cells is detected by a CCK-8 method, and the experimental result is analyzed to obtain the IC 50 . (C) The MDA-MB-231 breast cancer cells of the experimental group are induced by adding 9' -salvianolic acid B monomethyl ester (35 mu mol/L) for 24 hours, and the culture is continued for 12 hours after marking. The upper graph is left and right of scratches taken by the control group and the experimental group at 0h after the scratches, and the lower graph is left and right of scratches taken by the control group and the experimental group at 12h after the scratches. (D) MDA-MB-231 breast cancer cells are induced by adding 9' -salvianolic acid B monomethyl ester (35 mu mol/L) for 24 hours, and are continuously cultured for 12 hours after marking out traces, so that the trace healing rate is calculated, and differential statistics analysis is carried out. (E) After 9' -salvianolic acid B monomethyl ester (35 mu mol/L) is added to MDA-MB-231 breast cancer cells of an experimental group for 24 hours, the cells are planted in the upper chamber of the Boeden cell and are migrated for 6 hours. The left image is a picture of the chamber taken after 6h of migration of the control image. The right image is the real picture (F) of the small room taken after the experimental group is migrated for 6hThe experimental group MDA-MB-231 breast cancer cells are added with 9' -salvianolic acid B monomethyl ester (35 mu mol/L) to induce for 24 hours and then are planted in the upper chamber of the Boeden cell, and are migrated for 6 hours. Photographs were taken under a microscope, the number of cells passing through the cell was counted, and differential statistical analysis was performed. * P <0.05, < P < 0.01, < P)<0.001，****P<0.0001。
Detailed Description
The data processing and analysis methods of the following examples:
1. data processing
Boeden laboratory experiments, image J software was used to count the number of cells passing through the cell; scratch experiments, namely calculating scratch distances by using Image J software; MST experimental results were processed with MO. Affinity Analysis software. All pictures were plotted using GraphPad Prism 9 software.
2. Statistical analysis
The data variables were expressed as Mean ± standard error (Mean SEM) and statistical analysis was performed on the experimental data using Graph Pad Prism 9 software. Determining whether there is a statistical difference using a t-test for both sets of comparisons; analysis of variance was used for comparison between the groups to determine if the group-to-group differences were statistically significant. P <0.05 is the statistical difference between the data.
Example 1 micro thermal surge technique (MST)
Microphoresis technology, micro Scale Thermophoresis, abbreviated as MST technology. The Monolith series instrument quantitatively analyzes the intermolecular interactions by using a micro thermophoresis technique. One of the molecules is fluorescently labeled to become a reporter molecule, and the thermophoresis rule of the molecule in the solution is closely related to the molecular size, charge and hydration layer properties, so that MST signal change caused by combination is easily detected. The experimental results are automatically analyzed by software, so that the Kd value is accurately calculated.
1. First, samples of prepared His-Daam1 and His-FH2 proteins labeled with a fluorescent reagent (FH 2 is a domain of Daam1, FIG. 1) were pre-tested to detect fluorescence intensity, adhesion and uniformity of fluorescence intensity of the labeled proteins. The labeled protein sample was centrifuged well, and the supernatant was aspirated by a capillary tube and then detected by an upper machine.
2. The fluorescence intensity of the test result reaches more than 200, and the test result has no adsorption, no aggregation and good fluorescence uniformity, so that the next binding test can be performed. The binding experiment steps were as follows:
(1) mu.L of PBST was prepared, and two eight-way tubes, 10. Mu.L of PBST was added to each of the No. 2-16 tubes, and 20. Mu.L of lipond was added to the No. 1 tube.
(2) Taking 10 mu L of lipond out of the No. 1 tube, adding the lipond into the No. 2 tube, repeatedly blowing the solution uniformly by a pipetting gun, taking 10 mu L of lipond into the No. 3 tube, uniformly mixing the solution, diluting the No. 4-16 tube according to the same method, and finally discarding the redundant 10 mu L of lipond from the No. 16 tube.
(3) Adding 10 μl of labeled protein into each of the No. 1-16 tubes, mixing, centrifuging (10-15 min) the sample-added eight-joint tube, and detecting by capillary suction.
(4) And enabling MO. Affinity Analysis software to complete data Analysis and report export.
3. The results show (FIG. 2) that Daam1 full-length protein and 9 "-salvianolic acid B monomethyl ester can be directly combined; the 9 "-salvianolic acid B monomethyl ester binding site is the FH2 domain of Daam 1.
Example 2
1. Cell resuscitation, culture, passage and cryopreservation
1. Cell resuscitation
The ultraviolet light of the biosafety cabinet is opened in advance, and the culture medium to be used is preheated in a water bath kettle at 37 ℃. Wearing protective gloves, taking out MDA-MB-231 breast cancer cells to be revived from a liquid nitrogen tank or in the liquid nitrogen tank, rapidly putting into a water bath, and gently shaking until the cell suspension therein is completely melted. Spraying 75% alcohol to hands and the frozen tube, and placing into a biosafety cabinet. An equal amount of medium was added and then pipetted into a 15mL centrifuge tube. The centrifuge tube was placed in a centrifuge after marking the cell type and cell number, and centrifuged for 2min after balancing. Taking out after centrifugation, spraying alcohol, and putting into a biosafety cabinet. The supernatant was sucked off using an electric negative pressure suction apparatus, and the culture medium was added and sucked uniformly. Transferring to 6cm culture dish, repeatedly blowing and sucking for several times to make cell distribution uniform, and finally marking cell type and algebra on small dish.
2. Cell passaging and culturing
Cells were passaged when the density of cell attachment growth reached 80-90%. The biological safety cabinet ultraviolet light source is opened in advance to irradiate the safety cabinet for 20min, and the culture medium, 0.25% pancreatin and sterilized PBS to be used are preheated in advance in a water bath kettle at 37 ℃. Then the grown cells are taken from a cell incubator to be placed in a biosafety cabinet, the supernatant is sucked by an electric negative pressure suction device, PBS is added for three times to wash away residual dead cells, and finally 1mL of 0.25% pancreatin is added for digestion, so that the types of the cells are different and the digestion time is different according to the situation. It was observed under the mirror that the single cells had been transformed, i.e. digestion was complete. The cell suspension was transferred to a 15mL centrifuge tube. Centrifuging, removing supernatant, adding culture medium, blowing and sucking for multiple times, transferring to 6cm culture dish, repeatedly blowing and sucking for multiple times to make cell distribution uniform, and finally marking cell type and algebra on small dish.
3. Cell cryopreservation
And (3) operating according to the steps of cell passage, taking out the centrifuge tube after centrifugation, spraying alcohol, putting into a biosafety cabinet, sucking and removing the supernatant by using an electric negative pressure aspirator, and then adding 1mL of complete culture medium for blowing and sucking uniformly. Taking a corresponding number of cryopreservation tubes, adding 100-300 mu L of cell suspension into each tube according to the number of cells, supplementing the complete culture medium to 1mL, finally adding DMSO (10%), placing the cells into a cell cryopreservation box, preserving at-8 ℃ for 24 hours, and transferring the cells into liquid nitrogen.
EXAMPLE 3,
1. Cell proliferation assay
1. When MDA-MB-231 breast cancer cells in the petri dish grew to 80-90%, they were seeded into 96-well plates after digestion, and according to the appropriate number of plated cells, approximately 100. Mu.L of cell suspension per well, four wells per group. And attaching the equal cells to the wall and adding medicine after the equal cells grow to 80-90%. The drug concentration gradient of the group after 24h of drug adding treatment is 20 mu mol/L, 40 mu mol/L, 60 mu mol/L, 80 mu mol/L, the concentration gradient of the group after 48h is 20 mu mol/L, 25 mu mol/L, 30 mu mol/L, 35 mu mol/L, and the drug concentration of the group after 72h is 15 mu mol/L, 20 mu mol/L, 25 mu mol/L and 30 mu mol/L.
2. After the dosing treatment, the old drug-containing medium was aspirated, 10. Mu.L of CCK-8 enhancement solution was added per well, and since the amount of CCK-8 added per well was small, it was suggested to gently tap the plate to aid in mixing after the addition of the reagent, possibly because of errors due to sticking of the reagent to the walls of the wells, or to directly prepare the medium containing 10% CCK-8 enhancement solution, adding it as a replacement, 100. Mu.L per well. Care should be taken not to have bubbles so as not to affect the detection of absorbance.
3. Culturing in an incubator for 1h.
4. Absorbance was measured at 450nm with a spectrophotometer, and the result was stored and then treated with GraphPad Prism 9. If absorbance is not measured temporarily, 10. Mu.L of CCK-8 reaction terminator may be added to each well, and the wells may be kept at 4℃in the dark without changing absorbance within 7 days.
5. The calculation formula is as follows: cell inhibition ratio = [ (Ac-As) ]/[ (Ac-Ab) ] ×100%
As: absorbance of experimental wells (medium containing cells and drug to be tested, CCK-8, drug to be tested).
Ac: control wells (medium with cells and test drug, CCK-8, no test drug) absorbance.
Ab: absorbance of blank wells (medium without cells and drug to be tested, CCK-8).
6. The results showed (FIG. 3A-B), that IC50 (half inhibition concentration) was 39.44. Mu. Mol/L, 27.63. Mu. Mol/L and 19.31. Mu. Mol/L when 9 "-salvianolic acid B monomethyl ester was used for treating breast cancer MDA-MB-231 cells for 24h, 48h and 72h, respectively, and that 20-80. Mu. Mol/L9" -salvianolic acid B monomethyl ester had an inhibitory effect on proliferation of breast cancer MDA-MB-231 cells.
2. Cell scratch assay
1. After the cells grow to 80% -90%, adding 0.25% pancreatin to digest, and the digestion steps are detailed in the above. After digestion, the seeds were plated into 6-well plates.
2. After cells in the 6-well plate grow to 90%, the cells are transfected or treated with drugs for 24 hours according to the experimental requirements, three traces are gently scratched in each well by using a white fine gun head, the cells are washed twice by PBS, the dead cells are sucked and removed, and then a DMEM high sugar culture medium containing 10% FBS is added.
3. The photograph of the scratch is taken by a biological microscope, namely 0h, and the photograph is taken after 12h according to the growth speed of different cells, namely 12h.
4. Scratch distance differences or area differences were calculated with Image J and data were statistically analyzed for differences with GraphPad Prism 8.
5. The results showed (FIGS. 3C-D) that 35. Mu. Mol/L9 "-salvianolic acid B monomethyl ester significantly inhibited migration of breast cancer MDA-MB-231 cells.
3. Boiden laboratory experiment
1. After the cells grow to 80% -90%, adding 0.25% pancreatin to digest, centrifuging, discarding the upper layer culture medium, and seeding into the upper chamber of Boyden chamber (Boyden chamber) containing pre-spread matrigel.
2. After the cells had attached, the upper medium was aspirated, the cells were resuspended in medium without serum, the lower Boyden chamber (Boyden chamber) was filled with medium with 20% fetal bovine serum, the upper chamber was filled with 150. Mu.L of cell suspension and incubated for 6h.
After 3.6h, the cell upper and lower media were aspirated, washed several times with PBS, and then the lower cell was fixed with 600. Mu.L of 4% PFA for 20min. PFA was blotted off, washed several times with PBS, and stained with 600. Mu.L of crystal violet solution in the lower chamber for 20min.
4. After the dyeing is completed, the cell is gently taken out by forceps and cleaned, and then the upper cells are gently wiped off by a cotton swab.
5. Photographs of cells passing through the cells were taken with a microscope, and were taken in a "delta" shape.
6. Cell numbers were calculated with Image J and the resulting data were treated with GraphPad Prism 9.
7. The results showed (FIGS. 3E-F) that 35. Mu. Mol/L9 "-salvianolic acid B monomethyl ester significantly inhibited invasion of breast cancer MDA-MB-231 cells.
Claims (5)
1.9' -application of salvianolic acid B monomethyl ester in preparing medicines for treating breast cancer.
2.9' -application of salvianolic acid B monomethyl ester in preparing medicines for inhibiting breast cancer metastasis and/or invasion.
3.9' -application of salvianolic acid B monomethyl ester in preparing medicines for inhibiting breast cancer proliferation.
4. A pharmaceutical composition is characterized by containing 9' -salvianolic acid B monomethyl ester.
5. The pharmaceutical composition according to claim 4, further comprising pharmaceutical excipients required for preparing 9 "-salvianolic acid B monomethyl ester into pharmaceutical preparations.
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| CN106093223A (en) * | 2016-05-31 | 2016-11-09 | 天津中医药大学 | The research method of a kind of Radix Salviae Miltiorrhizae for injection Polyphenol Acids stability and the analysis method of degradation pathway thereof |
| US20210106552A1 (en) * | 2018-04-11 | 2021-04-15 | Howard University | Methods for treating triple negative breast cancer using salvianolic acid b |
| CN113791154A (en) * | 2021-09-17 | 2021-12-14 | 天津中医药大学 | Method for identifying chemical components in Xuebijing injection |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106093223A (en) * | 2016-05-31 | 2016-11-09 | 天津中医药大学 | The research method of a kind of Radix Salviae Miltiorrhizae for injection Polyphenol Acids stability and the analysis method of degradation pathway thereof |
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