CN117883420A - Application of pteris lobata extract - Google Patents
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Abstract
The invention discloses an application of a small-leaf Chinese fern extract, belonging to the technical field of biological medicine, wherein the extract is ent-8 (14), 15-pimaric diene-2 beta, 19-diol, and the cell proliferation of triple negative breast cancer MDA-MB-468 which is highly expressed by EGFR and the protein expression of a downstream signal path thereof is inhibited by reducing the EGFR, a colony formation experiment shows that the extract can obviously inhibit the clone formation of MDA-MB-468 so as to achieve the capacity of inhibiting the cell proliferation for a long time, and a cell scratch experiment proves that the extract also can inhibit the migration capacity of the triple negative breast cell MDA-MB-468 by regulating the EGFR related signal path, so that the extract has good breast cancer resisting potential; the compound is easy to prepare, and is further developed into a novel candidate medicament for clinical breast cancer treatment.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to application of a pteris lobata extract.
Background
ent-8 (14), 15-pimaradiene-2 beta, 19-diol (JXE-23) is a compound isolated from pteridophyte in China, and has been found from previous studies that JXE-23, together with other diterpenes, can significantly reduce the amount of NO production in cells and exert anti-inflammatory effects by blocking the expression and transcription of iNOS proteins and inflammatory mediators TNF-alpha and IL-6.
Triple negative breast cancer (Triple Negative Breast Cancer, TNBC) is a special subtype of breast cancer that is known for its strong aggressiveness, poor prognosis, limited clinical treatment options, and lack of effective specific targeted therapies. TNBC accounts for 15% -20% of breast cancers diagnosed, and is characterized by the lack of three specific receptors: estrogen Receptor (ER), progestin Receptor (PR) and human epidermal growth factor receptor 2 (HER 2). Treatment of such invasive diseases is challenging due to the lack of targeted receptors expressed in other types of breast cancer and the complex pathological processes involved. To date, nonspecific treatments such as surgery, conventional radiation and chemotherapy have been the only treatment options. Therefore, the individual differences of TNBC patients in the development of new TNBC treatment are obvious, clinical sample data are single, most of TNBC typing is based on mRNA levels of different genes, and the mRNA expression levels cannot accurately reflect the protein expression levels due to various modification and regulation steps in the translation process of proteins, so that the defects greatly influence the targeted treatment and postoperative prediction of patients. There is currently no effective targeted therapy for TNBC, and multiple treatments may be required to significantly improve patient prognosis. Chemotherapy is one of the main approaches to TNBC treatment, and is often clinically intervened by drug combination, and combination chemotherapy of taxane and anthracycline is the standard treatment for early-stage TNBC patients, but it increases to some extent the risk of cardiac mortality, secondary leukemia and myelodysplastic syndrome, carboplatin increases the incidence of neutropenia and thrombocytopenia, while bevacizumab causes hypertension, infection, thrombotic blockage, hemorrhage and postoperative complications. In order to develop a novel medicine for treating TNBC, the invention provides the following technical scheme.
Disclosure of Invention
The invention aims to provide an application of a pteris lobata extract, and provides a new idea and a new method for clinical TNBC treatment.
The aim of the invention can be achieved by the following technical scheme:
use of a fern extract as sole active ingredient in the manufacture of a medicament for the treatment and/or prophylaxis of triple negative breast cancer;
the extract of the pteris lobata is ent-8 (14), 15-pimarica-2 beta, 19-diol:
further, the drug is an inhibitor for inhibiting proliferation of cancer cells.
Further, the drug is an inhibitor for inhibiting cancer cell migration.
Further, the medicament is used for inhibiting EGFR/Akt/ERK/p70 S6K Signaling pathway to decrease the viability of MDA-MB-468 cells.
The invention has the beneficial effects that:
the compound ent-8 (14), 15-pimaric diene-2 beta, 19-diol is separated from the small leaf Chinese fern, and can obviously inhibit proliferation of breast cancer cells; scratch experiments show that the compound can inhibit the migration of TNBC cells with high migration capacity; further verifies that the compound inhibits EGFR/Akt/ERK/p70 by western blot experiment S6K The signaling pathway reduces MDA-MB-468 cell viability. The results show that the ent-8 (14), 15-pimaric diene-2 beta, 19-diol has better TNBC resistance and potential application value in the aspect of TNBC treatment in the future.
Drawings
The invention is further described below with reference to the accompanying drawings.
FIG. 1 is a schematic diagram showing the inhibition of TNBC cell growth by MTT assay test compound ent-8 (14), 15-pimaric diene-2 beta, 19-diol;
FIG. 2 is a graph showing the effect of compound ent-8 (14), 15-pimaric diene-2 beta, 19-diol on migration of TNBC cells MDA-MB-468 (scale: 100 μm,/p <0.001vs Con);
FIG. 3 is a schematic representation of the effect of compound ent-8 (14), 15-pimaric diene-2 beta, 19-diol on TNBC cell morphology of MDA-MB-468;
FIG. 4 shows the compounds ent-8 (14), 15-pimaric diene-2 beta, 19-diol pair EGFR and the downstream signaling pathway related proteins Akt, ERK and p70 S6K Schematic representation of the effect of expression.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
ent-8 (14), 15-pimpinene-2 beta, 19-diol is a compound separated from pteris lobata, and the structural formula of the compound is as follows:
example 1: determination of the growth inhibitory Effect of ent-8 (14), 15-pimaric diene-2 beta, 19-diol on cancer cells by MTT method
(1) Seeding cells
Taking cells with good growth state and up to 90% of survival rate for cell passage, collecting cell precipitate, preparing into single cell suspension, inoculating cells into 96-well plate according to proper cell density (1000-10000 cells/well), adding 100 μl single cell suspension into each well, arranging 3 multiple wells, observing cell state under mirror, and placing into a cell culture medium containing 5% CO 2 Incubator at 37℃with 5% CO 2 Cells were cultured for 24h under saturated humidity.
(2) Dosing treatment
CellsAfter the adherence is carried out for 24 hours, proper medicine concentration gradient is set according to the sensitivity degree of cells to medicines, medicine culture mediums with different concentrations are prepared according to a medicine formula, a 96-well plate is taken out, the supernatant is discarded, medicine culture mediums with different concentrations are sequentially added, 3 compound holes are arranged in each group, blank contrast is arranged, and after the cell state is observed under the lens, the cells are continuously placed in CO 2 Culturing in an incubator.
(3) MTT assay
After the drug is incubated for 70h, 10 mu L of MTT solution with the concentration of 5mg/mL is added into each hole, and the cells are continuously placed in CO after being observed under a lens 2 Culturing in an incubator for 2 hours; taking out the 96-well plate when MTT of the control group is fully combined with cells to form insoluble blue-violet crystal formazan, carefully discarding the solution of each well, adding 150 mu L of DMSO, and oscillating at a low speed for 5min to fully dissolve formazan in the cells; the microplate reader was turned on, the wavelength was set at 490nm, absorbance (OD) was read, data was saved, and each experiment was repeated three times independently.
(4) Data analysis
According to the formula: inhibition (%) = (control OD-experimental OD)/(control OD-blank OD) ×100 inhibition of cell growth at different drug concentrations was calculated and semi-inhibitory concentration (IC 50) of drug in cells was calculated using GraphPad Prism 8 software.
The results of screening for ent-8 (14), 15-pimaric diene-2 beta, 19-diol using the MTT method are shown in Table 1. In TNBC cells, the half inhibitory concentration of ent-8 (14), 15-pimaric diene-2 beta, 19-diol on MDA-MB-468 was minimal.
To further verify the growth inhibition of MDA-MB-468 cells by ent-8 (14), 15-pimaradiene-2 beta, 19-diol, the MTT assay was used for 24h,48h and 72 hen-8 (14), and the results of the inhibition of MDA-MB-468 by 15-pimaradiene-2 beta, 19-diol are shown in FIG. 1. The inhibition of MDA-MB-468 by ent-8 (14), 15-pimaradiene-2 beta, 19-diol was dose dependent.
TABLE 1 IC50 values (μM+ -SD) of 15-pimaric diene-2 beta, 19-diol for different TNBC cells (14)
Example 2: trypan blue staining counting method for detecting influence of compound ent-8 (14), 15-pimaric diene-2 beta, 19-diol on MDA-MB-468 cell proliferation
(1) Seeding cells
The cell sediment obtained by carrying out cell passage when the growth state is good and the cell growth density reaches 90 percent, centrifuging at 800rpm for 5min is resuspended by 1mL of culture medium to prepare single cell suspension, and the cell count is carried out according to the ratio of 3.5X10 5 Cell Density of individual/well cells were seeded into 12 well plates and observed under a microscope for cell status and then placed in a chamber containing 5% CO 2 Incubator at 37℃with 5% CO 2 Cells were cultured for 24h under saturated humidity.
(2) Dosing treatment
After cell adherence is carried out for 24 hours, proper drug concentration gradients of 2.5 mu M,5 mu M and 10 mu M are set according to half inhibition concentration of drugs, drug culture mediums with different concentrations are prepared, a 12-pore plate is taken out, the supernatant is discarded, the drug culture mediums with different concentrations are sequentially added, 3 compound holes are arranged at each concentration, blank control is arranged, and after cell state is observed under a lens, the cell culture mediums are continuously placed in CO 2 Culturing in an incubator.
(3) Trypan blue staining count detection
After incubation of the drug for 24h,48h,72h, respectively, cells were digested and resuspended in one set of concentration gradients, each concentration of cell suspension was diluted in PBS for three sets of replicates, respectively, stained with 0.4% trypan blue for 2min, counted separately under an inverted microscope with a hemocytometer, and recorded for 24h,48h,72h, respectively.
(4) Data analysis
Each concentration was counted 3 times independently and the results were plotted using GraphPad Prism 8.0.2 software and statistically analyzed to obtain a growth curve for cells after drug treatment.
Example 3: scratch assay to detect effects of compound ent-8 (14), 15-pimaric diene-2 beta, 19-diol on MDA-MB-468 cell migration
(1) Seeding cells
Three horizontal transverse lines are drawn on the bottom of a 6-hole plate by a marker pen in advanceTo ensure that the subsequent photographing positions are the same. Taking cells in logarithmic growth phase for cell passage, collecting cell precipitate, preparing into single cell suspension, counting, and mixing with appropriate cell density (MDA-MB-468:5.0X10) 5 A/hole; MDA-MB-468: 1.0X10 6 Single cell suspension of 2mL per well, observing cell state under a mirror after uniformly mixing by a cross method, and carrying out CO at 37 ℃ and 5 percent 2 Cells were cultured overnight under saturated humidity conditions.
(2) Scribing line
The next day, cells were observed under the microscope to be spread on the whole dish bottom (a monolayer of cells with 100% fusion) and a 200. Mu.L-sized pipette tip was prepared for streaking, the streaking track was kept perpendicular to the three horizontal transverse lines, streaking was performed from one end to the other end along the sterilized ruler, a clear streaking was observed on the cell surface at this time, the old medium was discarded, the cells which had fallen off when streaking was removed were washed with PBS multiple times, the cell state was observed under the microscope and photographed for storage, and the photographed area (area above or below the intersection of streaking and alignment) was recorded as a 0h control group for the next observation. Note that: the pipette tip is used for streaking, and the pipette tip is always perpendicular to the surface of the cell and is the same.
(3) Dosing treatment
To reduce false positive results caused by cell proliferation, reduce the effect of cell proliferation on experimental results, corresponding concentrations of drug (MDA-MB-468: 0. Mu.M, 2.5. Mu.M, 5. Mu.M, 10. Mu.M) were prepared in low serum medium (2% FBS), and after 72h differences in migration ability between the treatment group and the control group were observed and photographed.
(4) Optical microscope observation photographing
And (3) after 72h, observing the healing condition of the scratch, discarding the old culture medium, washing 3 times by using PBS, finding the marking position of the 0h control group, photographing and recording, and storing the picture in a Tif format.
(5) Data analysis
Scratch area was calculated with Image J software, cell mobility (%) = (control area-experimental area/control area) ×100, each experiment was independently repeated 3 times, and the results were plotted with GraphPad Prism 8 software and statistically analyzed.
As shown in fig. 2, the scratch width of the control group was gradually narrowed with the lapse of time, while the addition group significantly inhibited the healing of scratches with the increase of concentration at 72h, and was dose-dependent. The experimental result shows that the compound ent-8 (14), 15-pimaric diene-2 beta, 19-diol can obviously inhibit the migration of MDA-MB-468 cells.
Example 4: influence of Compound ent-8 (14), 15-pimaric diene-2 beta, 19-diol on morphological changes in MDA-MB-468 cells
(1) Seeding cells
The cell sediment obtained by carrying out cell passage when the growth state is good and the cell growth density reaches 90 percent, centrifuging at 800rpm for 5min is resuspended by 1mL of culture medium to prepare single cell suspension, and the cell count is carried out according to the ratio of 1.0X10 5 Cell Density of each well cells were seeded into 6 well plates and observed under a microscope for cell status and then placed in a 6 well plate containing 5% CO 2 Incubator at 37℃with 5% CO 2 Cells were cultured for 24h under saturated humidity.
(2) Dosing treatment
After cell adhesion for 24h, preparing medicine culture medium with medicine concentration gradient of 2.5 mu M,5 mu M and 10 mu M, taking out 6-hole plate, discarding supernatant, adding medicine culture medium with different concentrations in sequence, setting a group of non-medicine adding control group, observing cell state under mirror, and continuously placing in CO 2 Culturing in an incubator for 24 hours.
(3) Cell morphology observations
As shown in fig. 3, the 6-well plate after 24h of cell dosing treatment is taken out, the cell morphology is observed under an inverted phase contrast microscope, and a picture is taken to obtain a cell phase contrast morphology, then the 6-well plate is washed once with PBS, 1mL of methanol is fixed for 15min, the PBS is washed once again, the cells with the concentration of 0.005% are dyed for 20min, and the cells with each concentration are continuously taken under a common inverted microscope to obtain a crystal violet dyed cell morphology.
Example 5: western blotting method for detecting influence of compound ent-8 (14), 15-pimaric diene-2 beta, 19-diol on protein expression in MDA-MB-468 cells
(1) Seeding cells
Taking MDA-MB-468 cells with good growth state and 90% confluence for cell passage, collecting cell sediment, preparing into single cell suspension, counting cells, and obtaining the final product according to 1.0X10 5 Cell Density of each well cells were seeded into 6 well plates with 2mL single cell suspension per well, the cell state was observed under a "crisscross" method, shaking-in-a-mirror, at 37℃and 5% CO 2 Cells were cultured for 24h under saturated humidity.
(2) Dosing treatment
After 24h of cell adherence, preparing drug culture medium with drug concentration gradient of 0 μM,2.5 μM,5 μM and 10 μM, taking out 6-hole plate, discarding supernatant, sequentially adding the above drug culture medium with different concentrations, and continuously placing in CO 2 Culturing in an incubator for 24 hours.
(3) Cellular protein extraction
Observing the state of MDA-MB-468 cells after cell drug addition treatment under a lens, and adjusting the content of cell lysate according to the cell density of each hole. Placing a 6-hole plate on ice, collecting proteins, performing the whole process on the ice, discarding the supernatant, washing cells with precooled PBS, discarding the PBS, then uniformly dripping corresponding cell lysate, after full lysis, collecting the lysed cells in a precooled 1.5mL centrifuge tube by precooled and clean cell scrapers, respectively boiling for 4min in a water bath at 100 ℃ for a total time of not more than 10min, fully denaturing the proteins, and storing in a refrigerator at-20 ℃ after vortex mixing.
(4) Western blot detection
Protein samples were loaded on each well of a sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE), separated by electrophoresis, and transferred to NC membranes. After blocking with 5% (w/v) skimmed milk for 1h at room temperature, NC membrane containing protein was incubated with primary antibody to the corresponding protein, and slowly incubated overnight at 4℃with a rotary shaker. After four washes with TBS-T containing 0.05% (v/v) Tween-20, incubation with specific secondary antibodies was performed for 2h at room temperature. The membrane was then washed four more times with TBS-T. The immunoreactive bands were developed with an enhanced chemiluminescent substrate. An anti-beta-actin antibody was used as a control. Finally, the strip is straightened and cut by using PS software, the result is shown in fig. 4, the gray value of the strip is quantitatively analyzed by using Image J software, and normalization processing is carried out by using excel software.
The foregoing is merely illustrative and explanatory of the invention, as various modifications and additions may be made to the particular embodiments described, or in a similar manner, by those skilled in the art, without departing from the scope of the invention or exceeding the scope of the invention as defined in the claims.
Claims (4)
1. Use of a fern extract as sole active ingredient in the manufacture of a medicament for the treatment and/or prophylaxis of triple negative breast cancer;
the extract of the pteris lobata is ent-8 (14), 15-pimarica diene-2 beta, 19-diol, and the structural formula is as follows:
2. the use according to claim 1, wherein the medicament is an inhibitor of cancer cell proliferation.
3. The use according to claim 1, wherein the medicament is an inhibitor of cancer cell migration.
4. The use according to claim 1, wherein the medicament is for inhibiting EGFR and its downstream signaling pathway related proteins Akt, ERK and p70 S6K An inhibitor to reduce MDA-MB-468 cell viability.
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