CN114983988A - Application of teprenone in preparation of medicine for treating and/or preventing cancer - Google Patents
Application of teprenone in preparation of medicine for treating and/or preventing cancer Download PDFInfo
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- DJAHKBBSJCDSOZ-AJLBTXRUSA-N (5z,9e,13e)-6,10,14,18-tetramethylnonadeca-5,9,13,17-tetraen-2-one;(5e,9e,13e)-6,10,14,18-tetramethylnonadeca-5,9,13,17-tetraen-2-one Chemical compound CC(C)=CCC\C(C)=C\CC\C(C)=C\CC\C(C)=C/CCC(C)=O.CC(C)=CCC\C(C)=C\CC\C(C)=C\CC\C(C)=C\CCC(C)=O DJAHKBBSJCDSOZ-AJLBTXRUSA-N 0.000 title claims abstract description 141
- 229950006156 teprenone Drugs 0.000 title claims abstract description 105
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- -1 Tetraprenom Chemical compound 0.000 description 2
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 2
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- CDOSHBSSFJOMGT-JTQLQIEISA-N (R)-linalool Natural products CC(C)=CCC[C@@](C)(O)C=C CDOSHBSSFJOMGT-JTQLQIEISA-N 0.000 description 1
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- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
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- 208000003721 Triple Negative Breast Neoplasms Diseases 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- SMZOGRDCAXLAAR-UHFFFAOYSA-N aluminium isopropoxide Chemical compound [Al+3].CC(C)[O-].CC(C)[O-].CC(C)[O-] SMZOGRDCAXLAAR-UHFFFAOYSA-N 0.000 description 1
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- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical class O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 1
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Images
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/12—Ketones
- A61K31/121—Ketones acyclic
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/04—Antineoplastic agents specific for metastasis
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- Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Oncology (AREA)
- Epidemiology (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
Abstract
Use of teprenone in the manufacture of a medicament for the treatment and/or prevention of cancer. The experimental data of the invention show that the all-trans teprenone has the effect obviously superior to that of the mixed teprenone in the aspects of inhibiting the proliferation of breast cancer cells, non-small cell lung cancer cells, lung adenocarcinoma cells and colon cancer cells, has low toxicity in human embryonic kidney cells, has the effect superior to that of the mixed teprenone in the migration of the breast cancer cells, and reveals that the all-trans teprenone can be used for preparing the medicines for treating and/or preventing the diseases.
Description
Technical Field
The invention relates to the field of biomedicine, in particular to application of teprenone in preparing a medicament for treating and/or preventing cancer.
Background
According to the latest global cancer burden data published by the international agency for research on cancer (IARC) of the world health organization in 2020, worldwide colorectal cancer becomes the third most common cancer pattern second only to lung cancer.
According to the american cancer society, about 26 million women are diagnosed with breast cancer each year, with 15% to 20% of them having triple negative breast cancer. The incidence of young breast cancer patients worldwide shows a significantly rising trend. In the united states, about 11% of breast cancer patients are between 35 and 45 years of age, while the rate of young breast cancer patients in the asian population is significantly higher than in the western world, accounting for 9.5% to 12% of all asian breast cancer patients.
The lung cancer is a malignant tumor with the highest global and national mortality rate, the non-small cell lung cancer accounts for more than 85% of all lung cancer cases, and if the lung cancer is not treated in time, the median survival time is only 4-5 months. Approximately 70% to 80% of patients have been diagnosed at a middle or advanced stage, and therefore, non-surgical treatment modalities play an important role for most lung cancer patients.
Cytotoxic drug chemotherapy is currently an effective treatment option for this group of people, but the overall survival time (O S) of patients is only about 12 months, and the 3-year and 5-year survival rates of patients are only 19% and 11%, respectively.
Disclosure of Invention
In one embodiment, the application of teprenone in preparing a medicament for treating and/or preventing breast cancer, non-small cell lung cancer, lung adenocarcinoma and colon cancer is provided.
In one embodiment, the teprenone is at least one of all-trans teprenone and single-cis teprenone.
In one embodiment, the teprenone is all-trans teprenone.
In one embodiment, the teprenone is mono-cis-teprenone.
In one embodiment, the teprenone comprises all-trans teprenone, single cis-teprenone.
In one embodiment, the teprenone comprises all-trans teprenone, single-cis teprenone, and the molar ratio of all-trans teprenone: mono-cis-teprenone ═ (0.6-0.7): 1.
in one embodiment, the teprenone is used to inhibit proliferation and/or migration of human breast cancer cells.
In one embodiment, the teprenone is used to inhibit proliferation of human non-small cell lung cancer cells.
In one embodiment, the teprenone is used to inhibit proliferation of human lung adenocarcinoma cells.
In one embodiment, the teprenone is used to inhibit human colon cancer cell proliferation.
In one embodiment, the teprenone is used to inhibit proliferation of human rectal cancer cells.
In one embodiment, the teprenone has low toxicity to human normal cells.
In one embodiment, the IC of teprenone in human normal cells 50 ＞10μmol/L。
In one embodiment, the human normal cells comprise human embryonic kidney cells.
According to the application of the teprenone in the preparation of the medicine for treating and/or preventing the cancer, the experimental data of the invention show that the all-trans teprenone has the effect of obviously superior to that of mixed teprenone in the aspects of inhibiting the proliferation of breast cancer cells, non-small cell lung cancer cells, lung adenocarcinoma cells and colon cancer cells.
In one embodiment, the all-trans teprenone has low toxicity in human embryonic kidney cells, and the all-trans teprenone has better effect than mixed teprenone in the inhibition of breast cancer cell migration, and the all-trans teprenone can be used for preparing the medicine for treating and/or preventing the diseases.
Drawings
FIG. 1 is a graph showing the results of an experiment for inhibiting the proliferation of human breast cancer cells;
FIG. 2 is a graph showing the results of the proliferation inhibition experiment for human breast cancer cells at different times;
FIG. 3 is a graph showing the results of the scratch test;
FIG. 4 is a graph showing the results of a migration experiment;
FIG. 5 is a graph showing the results of an experiment for inhibiting proliferation of human NSCLC cells;
FIG. 6 is a graph showing the results of a human lung adenocarcinoma cell proliferation inhibition experiment;
FIG. 7 is a graph showing the results of an experiment for inhibiting the proliferation of human colon cancer cells;
FIG. 8 is a graph showing the results of an experiment for inhibiting the proliferation of human colon cancer cells;
FIG. 9 is a graph showing the results of the human embryonic kidney cell proliferation inhibition experiment.
Detailed Description
The present invention will be described in further detail with reference to the following detailed description and accompanying drawings. In the following description, numerous details are set forth in order to provide a better understanding of the present application. However, one skilled in the art would readily recognize that some of the features may be omitted in different instances or may be replaced by other materials, methods. In some instances, certain operations related to the present application have not been shown or described in detail in order to avoid obscuring the core of the present application from excessive description, and it is not necessary for those skilled in the art to describe these operations in detail, so that they may be fully understood from the description in the specification and the general knowledge in the art.
Furthermore, the features, operations, or characteristics described in the specification may be combined in any suitable manner to form various embodiments. Also, the various steps or actions in the description of the methods may be transposed or transposed in order, as will be apparent to a person skilled in the art. Thus, the various sequences in the specification and drawings are for the purpose of describing certain embodiments only and are not intended to imply a required sequence unless otherwise indicated where such sequence must be followed.
The numbering of the components as such, e.g., "first", "second", etc., is used herein only to distinguish the objects as described, and does not have any sequential or technical meaning.
Herein, "μmol/L" and "μ M" are used interchangeably.
IC 50 The median inhibitory concentration, refers to in vitro experiments (in vitro), after a specified exposure time. The concentration of drug required for 50% inhibition of the cells. The dose-response curve (dose-response curve) can be obtained by calculating the successive cytostatic rates of different concentrations according to the following formula, and the IC can be estimated 50 The value is obtained. IC (integrated circuit) 50 The smaller the size, the stronger the effect of the drug.
cell inhibition＝1-cell viability
Wherein cell viability refers to cell viability;
OD drug (experimental group) represents the absorbance of the wells containing cells at a particular dosing concentration;
OD control (negative control group) represents the absorbance of the wells containing cells at the administration concentration of 0, taking the cell line viability of this group as 100% in the experiment;
OD blank (blank) represents absorbance of wells without cells for zero adjustment of absorbance;
cell inhibition refers to the rate of cell inhibition.
IC 50 I.e., the dose concentration corresponding to the cell inhibition rate (cell inhibition) of 50%.
While IC 50 Calculated by the following formula:
wherein OD drug-T Represents the absorbance of the cell-containing wells at the time T of drug exposure in the experimental group.
OD control-T (negative)Control group) represents the absorbance of the wells containing cells at the dose concentration of 0 and the exposure time of T.
The commercial name of teprenone is Schvesuk, which is a gastric mucosa protective agent developed by Euonymus japonicus Kabushiki Kaisha, is marketed in Japan in 1984 and approved by FDA in 1994, and is clinically used for treating acute and chronic gastritis and gastric ulcer. Also known as pentaerythrenone, E-671, Selbex, Tetraprenom, Tetraprenylacetone, Tetraprenone. Teprenone (GGA), a mixture of all-trans and mono-cis isomers of 6,10,14, 18-tetramethyl-5, 9,13,17-nonadecatetraen-2-one (molar ratio 3: 2), is an acyclic isoprenoid compound having a retinoid backbone.
At present, teprenone is mainly used for preparing medicines for preventing and treating ovarian cancer, glaucoma and depression, treating opioid addiction and preventing relapse, preparing medicines for preventing and treating liver injury, virus infection, fatty liver and steatohepatitis caused by morphine and preventing and treating toxic hepatitis and the like.
In one embodiment, the application of teprenone in preparing a medicament for treating and/or preventing breast cancer, non-small cell lung cancer, lung adenocarcinoma, colorectal cancer and embryonic renal cell tumor is provided.
In one embodiment, the teprenone is at least one of all-trans teprenone and single-cis teprenone.
In one embodiment, the teprenone is all-trans teprenone.
In one embodiment, the molar content of all-trans teprenone in the teprenone is greater than or equal to 98%.
In one embodiment, the teprenone is mono-cis-teprenone.
In one embodiment, the teprenone comprises all-trans teprenone, single-cis teprenone.
In one embodiment, the teprenone comprises all-trans teprenone, single-cis teprenone, and the ratio of single-cis teprenone: all-trans teprenone (0.6-0.7): 1, preferably (0.61-0.68): 1, more preferably 2: 3.
in one embodiment, the teprenone is used for inhibiting proliferation of human breast cancer cells.
In one embodiment, the teprenone is used to inhibit migration of human breast cancer cells.
In one embodiment, the teprenone is used for inhibiting proliferation of human non-small cell lung cancer cells.
In one embodiment, the teprenone is used to inhibit proliferation of human lung adenocarcinoma cells.
In one embodiment, the teprenone is used to inhibit human colon cancer cell proliferation.
In one embodiment, the teprenone is used to inhibit proliferation of human rectal cancer cells.
In one embodiment, the teprenone has low toxicity to human normal cells.
In one embodiment, the IC of teprenone in human normal cells 50 ＞10μmol/L。
In one embodiment, the human normal cells comprise human embryonic kidney cells.
In the following examples, the effects of proliferation inhibition and cell migration of all-trans teprenone (5E-GGA, labeled 2), single-cis teprenone (5Z-GGA, labeled 3) and a mixture of both (GGA, labeled 1) in breast cancer cells (MDA-MB-231) and in human non-small cell lung cancer cell line cells (A549), human lung adenocarcinoma cells (PC-9), and human colon cancer cells (KM12, HCT116), including specifically IC50 data, were tested primarily, and the results of the study show that all-trans teprenone is more IC than single-cis teprenone and the mixture 50 About 2 to 3 times lower. The toxicity effect of all-trans teprenone (5E-GGA, marked as 2), single-cis teprenone (5Z-GGA, marked as 3) and a mixture (GGA, marked as 1) of the two on human embryonic kidney cells (293T) is tested, and the result shows that the all-trans teprenone has low toxicity on the human embryonic kidney cells.
1. The preparation method of the compound comprises the following steps: preparation of all-trans teprenone (5E-GGA) and single-cis teprenone (5Z-GGA)
The reaction formula for preparing the teprenone mixture is as follows:
2. the experimental steps are as follows:
(1) into a reaction flask were added geranylaryl linalool (1.45g, 5mmol), methyl acetoacetate (5.8g, 50mmol) and aluminum isopropoxide (0.1g, 0.5mmol 1).
(2) The temperature is raised to 160 ℃ and stirred and heated for 6 hours. TLC detection of the disappearance of the starting material, cooling the reaction mixture to room temperature, diluting with 50mL of n-hexane, washing 3 times (20 mL. times.3) with 10% dilute sulfuric acid, separating the organic phase and washing with 20mL of saturated brine.
(3) Drying with anhydrous sodium sulfate, filtering, concentrating, and subjecting the residue to silica gel column chromatography with petroleum ether/ethyl acetate mixture as eluent, petroleum ether: ethyl acetate 100: 1, collecting each elution component, and concentrating by rotary evaporation.
(4) To give 0.51g of all-trans teprenone as a colorless transparent liquid with a purity of 99.5%, (Rf 0.6, PE/EA 50:1), ESI-MS M/z 331.27[ M + H ] +,353.24[ M + Na ] +.1H NMR (400MHz, CDC13) δ 5.09(M,4H),2.45 (M,2H),2.26(M,2H),2.14(s,3H),2.01(M,12H),1.68(s,3H),1.60(s, 12H); 0.2g of single-cis-teprenone is obtained, and the purity of the single-cis-teprenone is 98.1 percent, wherein (Rf is 0.65, PE/EA is 50:1) ESI-MS M/z is 331.27[ M + H ] +,353.24[ M + Na ] +, 1HNMR (400MHz, CDC13) delta 5.05-5.13 (M,4H), 2.42-2.47 (M,2H), 2.23-2.29 (M,2H),2.13(s,2H), 2.08-2.03 (t,8H), 1.96-1.99 (t,5H),1.68(s,4H), 1.57-1.61 (t, 11H).
In the following examples, the teprenone mixture was purchased from Rianlong (Tianjin) industries, Ltd., as a colorless transparent liquid with a purity of 99.5% by mol, 5E-GGA: 5Z-GGA: 3: 2.
Specific information for the ex-purebred mixture of teprenones is as follows:
the chemical structural formula of the teprenone is as follows:
the basic information is as follows:
the product name is as follows: teprenone;
CAS NO：6809-52-5；
chinese alias: 6,10,14, 18-tetramethyl-5, 9,13, 17-nonadecatetraen-2-one;
the molecular formula is as follows: c 23 H 38 O；
Molecular weight: 330.55, respectively;
english name: 5,9,13, 17-nonadecacatalen-2-one, 6,10,14, 18-tetramethyl-;
density: 0.872g/cm 3 ；
Flash point: 168 ℃;
boiling point: 442.2 ℃ at 760 mmHg.
Examples of biological activity assays are as follows:
example 1 MDA-MB-231 human breast cancer cells, the bioactivity test is specifically a proliferation inhibition test in MDA-MB-231 cells, and the detection method is a chemiluminescence method.
1) Method for dilution of compound: an appropriate amount of sample was weighed, dissolved in DMSO to a concentration of 20mmol/L, and diluted 20-fold with complete medium to give a final concentration of 1mmol/L of compound in the dilution. The assay was repeated with two-fold dilution, eight concentration gradients, and three replicates per well.
2) Culture of cells
2.1) preparation of complete medium: 1% double antibody and 10% inactivated fetal calf serum are added into the L-15 culture medium.
2.2) culture of cells: MDA-MB-231 cells were inoculated into culture medium and incubated at 37 ℃ with 0% CO 2 Culturing in an incubator, and carrying out passage for 2-3 days.
3) Determination of the inhibitory Effect of samples on the growth of MDA-MB-231 cells
Digesting the cells with EDTA-pancreatin digestive juice, centrifuging for 5min at 1000r, discarding the supernatant, resuspending with culture medium, diluting 100 μ L of the resuspended cell solution by 10 times, mixing, counting with a handheld cell counter, and culturingThe nutrient medium is diluted to 8x10 4 Each cell/mL, 100. mu.L/well was added to a 96-well plate, which was left at 37 ℃ with 0% CO 2 An incubator. After 24h of culture, 25. mu.L of diluted sample is added to each well, the initial concentration is 200. mu. mol/L, the culture box is placed, after 48h, 80. mu.L of chemiluminescent reagent is added to each well, the mixture is uniformly mixed after 10min of light shielding, and 140. mu.L of chemiluminescent reagent is absorbed from each well and added to a new 96-well white plate. And detecting by a chemiluminescence method of a microplate reader within 1 h. The IC can be calculated by using Prism 7 software after calculating the inhibition rates of different concentrations by using the inhibition rate calculation formula and using the cell without the sample and with the same DMSO concentration as the positive control and the complete culture medium as the negative control under the same condition 50 。
IC of sample Compounds in MDA-MB-231 cells 50 The results are shown in fig. 1 and table 1 below.
The inhibition rate calculation formula is as follows:
the inhibition rate was 100%, (luminescence signal intensity of positive control cells-luminescence signal intensity of experimental well cells)/(luminescence signal intensity of positive control cells-luminescence signal intensity of negative control cells) ].
Positive control: cells without sample and at the same concentration of DMSO were used as positive controls under the same conditions.
Experiment hole: containing compounds at different concentrations.
Negative control: complete medium was used as negative control.
TABLE 1 IC in human Breast cancer cells 50
| MDA-MB-231 | 1 | 2 | 3 |
| IC 50 | 117.9μM | 27.38μM | 54.03μM |
| R 2 | 0.9912 | 0.9947 | 0.9882 |
As can be seen, the IC of all-trans teprenone in the human breast cancer cell inhibition experiment 50 Minimum, 27.38 μ M; followed by mono-cis-teprenone, its IC 50 54.03 μ M; again, the mixture teprenone, its IC 50 The concentration was 117.9. mu.M. It can be deduced that all-trans teprenone plays a major inhibitory role in human breast cancer cells.
1) The dilution method of the compound is as follows: an appropriate amount of the sample was weighed, dissolved in DMSO to a concentration of 20mmol/L, and diluted 40-fold with complete medium to a concentration of 0.5 mmol/L. The assay was repeated with two-fold dilution, four concentration gradients, four per well.
2) Culture of cells
2.1) preparation of complete culture medium: 2% inactivated fetal bovine serum was added to the L-15 medium.
2.2) culture of cells: MDA-MB-231 cells were inoculated into L-15 medium containing 1% double antibody and 10% inactivated fetal bovine serum, and placed at 37 ℃ with 0% CO 2 Culturing in an incubator, and carrying out passage for 2-3 d.
3) Determination of the proliferation-inhibiting Effect of a sample on the growth of MDA-MB-231 cells
Digesting the cells with EDTA-pancreatin digestive juice, centrifuging for 5min at 1000r, discarding the supernatant, resuspending with complete culture medium, diluting 100 μ L of the resuspended cell solution by 10 times, mixing, counting with a handheld cell counter, and counting after the cell is used upDiluting the whole culture medium to 8 × 10 4 Each cell/mL, 100. mu.L/well was added to a 96-well plate, which was left at 37 ℃ with 0% CO 2 An incubator. After 4h of culture, 25 μ L of diluted sample is added to each well, the initial concentration is 100 μmol/L, the culture box is placed, 80 μ L of chemiluminescent reagent is added to each well after 48h and 72h respectively, the mixture is uniformly mixed after 10min of light shielding, and 140 μ L of chemiluminescent reagent is absorbed by each well and added to a new 96-well white board. And detecting by a chemiluminescence method of a microplate reader within 1 h. The proliferation inhibition of the sample compounds in MDA-MB-231 cells was obtained under the same conditions using cells without sample, with the same concentration of DMSO as a positive control and complete medium as a negative control.
The results are shown in FIG. 2. It can be seen that when the number of cells is 8000 per well, the all-trans teprenone is detected by chemiluminescence after being added for 48 hours, the inhibition effect is particularly obvious when the concentration of the all-trans teprenone is 50 mu mol/L, and the inhibition effect is more obvious after 72 hours.
At a concentration of 100. mu. mol/L and a time of 48h, the inhibitory effect of all-trans teprenone was the best, followed by the mixture, followed by the mono-cis teprenone.
At a concentration of 100. mu. mol/L and a time of 72h, the inhibitory effect of all-trans teprenone was the best, followed by the mixture, followed by the mono-cis teprenone.
Therefore, the concentration is 100 mu mol/L, the inhibition effect of all-trans teprenone, single-cis teprenone and the mixture is obvious, and the inhibition effect of all-trans teprenone is optimal.
Example 2 MDA-MB-231 human breast cancer cells, the bioactivity test is specifically a scratch migration inhibition test in MDA-MB-231 cells, and the detection method is a microscopic picture post-treatment.
The experimental steps are as follows:
1) method for dilution of compound: an appropriate amount of sample was weighed, dissolved in DMSO so that the final concentration of the compound in the dilution was 20mmol/L, and 6. mu.L of the sample solution (20mmol/L in DMSO) and 994. mu.L of complete medium were mixed.
2) Culture of cells
2.1) preparation of complete medium: 1% double antibody was added to the L-15 medium.
2.2) culture of cells: MDA-MB-231 cells were inoculated into L-15 medium containing 1% double antibody and 10% FBS, and incubated at 37 ℃ with 0% CO 2 Culturing in an incubator, and carrying out passage for 2-3 d.
3) Determination of scratch migration inhibition of samples on MDA-MB-231 cell growth
Taking a sterile disposable 6-hole plate, and uniformly marking 2 transverse lines on the back of the 6-hole plate by using a measuring ruler with a thin-head mark pen at intervals of about 7 mm; digesting cells with EDTA-pancreatin digestive juice, centrifuging for 5min at 1000r, discarding supernatant, resuspending with L-15 culture medium containing 1% double antibody and 10% FBS, diluting 100 μ L of the resuspended cell solution by 10 times, mixing, counting with a handheld cell counter, diluting with L-15 culture medium containing 1% double antibody and 10% FBS, adding 3mL of cell solution into a hole on a 6-well plate, and calculating MDA-MB-231 cells by 2 × 10 6 One/well, placed at 37 ℃ with 0% CO 2 An incubator. After 48 hours of culture, 3 vertical lines are vertically drawn in a 6-well plate with a measuring ruler by using a 300-mu-L clean gun head at intervals of about 7 mm. From left to right, positions 1-6 are marked from top to bottom. After the scratch was completed, the nonadherent cells were washed away using PBS rinse (3 mL. times.3) to make the gap left after streaking clearly visible, and then complete medium (2.5 mL/well) was added. Add 500. mu.L of the diluted compound solution to a 6-well plate and gently shake. Placing at 37 ℃ and 0% CO 2 The photo was taken at 0h and 24h of the cultivation in the incubator. The results of the inhibition of the scratch migration of the compounds in MDA-MB-231 cells were obtained under the same conditions using cells without sample and with the same concentration of DMSO as a positive control.
Table 2 scratch test mobility
| Time | Mobility ratio |
| 1-24h | 40.3％ |
| 2-24h | 42.6％ |
| 3-24h | 56.0％ |
| DMSO-24h | 46.4％ |
The mobility calculation formula is as follows: 24h mobility ═ a (0h) -a (24h))/a (0 h). Calculated using ImageJ software. A (0h) and A (24h) indicate the areas occupied by the cells in the cell scratch test at 0h and 24h, respectively.
As shown in fig. 3 and table 2, it can be seen that the mobility of the mixed (40.3%) and all-trans (42.6%) teprenone is lower than that of the positive (46.4%) control, while the mobility of the cis (56.0%) teprenone is higher than that of the positive (46.4%) control, indicating that all-trans teprenone in the teprenone mixture plays a major role in inhibiting migration.
Example 3 MDA-MB-231 human breast cancer cells, the bioactivity test is specifically a migration inhibition test in MDA-MB-231 cells, and the detection method is a crystal violet staining method.
The experimental steps are as follows:
1) method of dilution of the compounds: an appropriate amount of the sample was weighed, dissolved with DMSO so that the final concentration of the compound in the dilution was 20mmol/L, and diluted 100-fold and 200-fold with complete medium, respectively, so that the final concentration of the compound in the dilution was 0.2mmol/L and 0.1 mmol/L.
2) Culture of cells
2.1) preparation of complete medium: 0.1% BSA, 1% double antibody were added to the L-15 medium.
2.2) culture of cells: cells were cultured in complete medium for 24h (37 ℃, 0%CO 2 )。
3) Determination of migration inhibition of samples on MDA-MB-231 cell growth
Digesting the cells with EDTA-pancreatin digestive juice, centrifuging for 5min at 1000r, discarding the supernatant, resuspending with culture medium, diluting 100 μ L of the resuspended cell solution by 10 times, mixing, counting with a handheld cell counter, and diluting to 5 × 10 with culture medium 5 For each cell/mL, 600. mu.L of L-15 medium containing 10% FBS and 1% PS was added to the bottom of a 24-well plate, and 100. mu.L of the cell suspension was taken and added to a transwell chamber. Preparing a solution with a working concentration of 100 mu mol/L: 100 μ L (0.2mmol/L) of the compound solution was taken into each transwell cell; preparation of a solution with a working concentration of 50 mu mol/L: a solution of 100. mu.L (0.1mmol/L) of the compound was taken into each transwell chamber. Standing at 37 deg.C, with 0% CO 2 An incubator. After 48h, the transwell chamber was removed, the medium from the wells was discarded, rinsed 2 times with calcium-free PBS, and fixed for 30min by adding methanol (1 mL). The transwell chamber was removed, the wells were rinsed 2 times with PBS, 0.1% crystal violet (1mL) was added and stained for 20min, the upper non-migrating cells were gently wiped off with a cotton swab, the excess crystal violet solution in the wells was discarded, and rinsed 2 times with PBS. Preparation of 0.1% crystal violet: taking 1% crystal violet mother liquor (1mL), adding methanol (1mL), adding normal saline (8mL), and mixing well to prepare the product for use. The transwell chamber was removed, decolorized with 30% acetic acid (600. mu.L) for 10min, crystal violet was detached, 200. mu.L/well was added to a 96-well plate, and the OD570 value was measured with a microplate reader. Migration inhibition profiles of sample compounds in MDA-MB-231 cells were obtained under the same conditions using cells without sample, with the same concentration of DMSO as a positive control and complete medium as a negative control, and the results are shown in FIG. 4.
Therefore, the migration inhibition effect of all-trans teprenone and single-cis teprenone in 48h is obviously better than that of negative control and mixed teprenone, wherein the migration inhibition effect of all-trans teprenone is better than that of single-cis teprenone.
Example 4, A549 human non-small cell lung cancer cells, the bioactivity test is specifically a proliferation inhibition test in A549 cells, the detection method is CCK-8.
The experimental steps are as follows:
1. method of dilution of the compounds: an appropriate amount of sample was weighed, dissolved in DMSO so that the final concentration of the compound in the dilution was 20mmol/L, and diluted 20-fold with complete medium so that the final concentration of the compound in the dilution was 1 mmol/L. The assay was repeated with two-fold dilution, eight concentration gradients, two per well.
2. Cell culture
2.1 preparation of complete medium: 1% double antibody and 10% inactivated fetal bovine serum are added into the RPMI 1640 culture medium.
2.2 culture of cells: inoculating A549 cells into culture medium, standing at 37 deg.C and 5% CO 2 Culturing in an incubator, and carrying out passage for 2-3 d.
3. Determining the inhibition of A549 cell growth by the sample
3.1 collecting A549 cells, suspending after centrifugation, adding 5mL of RPMI 1640 culture medium for dilution, and counting the cells. 2000 cells per well, 2X 10 5 The cells were diluted in the loading chamber with RPMI 1640 medium to a final volume of 8 mL.
3.2 taking 96-well standard plate, marking positive control well, experimental well and blank well.
3.3 Add 80. mu.L of cell dilution to each of the positive control and experimental wells, 100. mu.L of medium to each of the blank wells, 100. mu.L of PBS to each of the peripheral wells of the well plate to prevent edge effect, incubate for 24 h.
3.4 starting concentration of compound 20mM, 10. mu.L was added to 190. mu.L of RPMI 1640 medium and diluted in a 96-well V-bottom plate with 8 gradients in 2-fold gradient (60. mu.L of diluted sample + 60. mu.L of RPMI 1640 medium).
3.5 transfer the compounds to the well plated cells, experiment wells 20. mu.L per well, positive control wells 20. mu.L of RPMI 1640 medium per well, incubator incubation 72 h.
3.6 Add 2mL of CCK-8 reagent to the wells and add 10. mu.L of CCK-8 reagent to each well.
Incubate at 3.737 deg.C in carbon dioxide incubator for 1 h.
Absorbance was measured at 450nM with a microplate reader of 3.8, and the final value was 450nM to 630nM absorbance using 630nM as a reference.
3.9 inhibition at various concentrations was calculated using inhibition calculation formula and IC was calculated using Prism 7 software 50 。
4. The results of the experiment are shown in fig. 5 and table 3.
TABLE 3 IC in human non-small cell Lung cancer cells 50
| |
1 | 2 | 3 |
| OD top | 84.4083 | 91.6299 | 82.8389 |
| OD Bottom | -10.3557 | 4.313 | 0.3557 |
| IC 50 | ～98.64μmol/L | 40.34μmol/L | 94.51μmol/L |
| R 2 | 0.984 | 0.9901 | 0.9976 |
As can be seen, the IC of all-trans teprenone in human non-small cell lung carcinoma cells (A549) 50 Minimum, 40.34 μmol/L; followed by mono-cis-teprenone, its IC 50 94.51 mu mol/L; and secondly the mixture teprenone, its IC 50 About 98.64. mu. mol/L. It can be concluded that all-trans teprenone plays a major inhibitory role in the proliferation of human non-small cell lung adenocarcinoma cells.
Example 5, PC-9 human Lung adenocarcinoma cells, the bioactivity test is specifically a proliferation inhibition test in PC-9 cells, the detection method is CCK-8.
The experimental steps are as follows:
1. method of dilution of the compounds: an appropriate amount of sample was weighed, dissolved in DMSO so that the final concentration of the compound in the diluent was 20mmol/L, and diluted 20-fold with complete medium so that the concentration was 1 mmol/L. The assay was repeated with two-fold dilution, eight concentration gradients, two per well.
2. Cell culture
2.1 preparation of complete medium: DMEM medium was supplemented with 1% double antibody, 10% inactivated fetal bovine serum.
2.2 culture of cells: inoculating A549 cells into culture medium, standing at 37 deg.C and 5% CO 2 Culturing in an incubator, and carrying out passage for 2-3 d.
3. Determination of the inhibitory Effect of samples on the growth of PC-9 cells
3.1 collect PC-9 cells, centrifuge, resuspend, add 5mL DMEM medium to dilute, count cells. 2000 cells per well, 2X 10 5 The cells were diluted in DMEM medium in the sample addition chamber to a final volume of 8 mL.
3.2 taking 96-well standard plate, marking positive control well, experimental well and blank well.
3.3 Add 80ul cell dilution to each well of positive control and experimental wells, 100 ul culture medium to each well of blank well, 100 ul PBS to each well of well plate periphery to prevent edge effect, incubate 24h in incubator.
3.4 initial concentration of compound 20mM, 10. mu.L was added to 190ul DMEM medium and diluted in 8 gradients in a 2-fold gradient in 96 well V-bottom plates (60. mu.L of diluted sample +60ul DMEM medium).
3.5 transfer the compounds to the plated cells, 20. mu.L per well of experimental wells, 20. mu.L of DMEM medium per well of positive control wells, and incubate for 72 h.
3.6 Add 2ml of CCK-8 reagent to the wells and add 10. mu.L of CCK-8 reagent to each well.
Incubate at 3.737 deg.C in carbon dioxide incubator for 1 h.
Absorbance was measured at 450nM with a microplate reader of 3.8, and the final value was 450nM to 630nM absorbance using 630nM as a reference.
3.9 inhibition at various concentrations was calculated using inhibition calculation formula and IC was calculated using Prism 7 software 50 。
4. The results of the experiments are shown in fig. 6 and table 4.
TABLE 4 human Lung adenocarcinoma cell IC 50
| |
1 | 2 | 3 |
| OD top | 90.1183 | 97.2615 | 96.5519 |
| OD Bottom | 2.9142 | 0.2200 | 3.4036 |
| IC 50 | 143.1000μmol/L | 51.9μmol/L | 53.6900μmol/L |
| R 2 | 0.9972 | 0.9839 | 0.9920 |
It can be seen that the IC of all-trans teprenone in human lung adenocarcinoma cells (PC-9) 50 The minimum is 51.9 mu mol/L; followed by mono-cis-teprenone, its IC 50 53.6 mu mol/L; again mixed teprenone, its IC 50 It was 143.1. mu. mol/L. It can be concluded that all-trans teprenone plays a major inhibitory role in human lung adenocarcinoma cell proliferation.
Example 6, KM12 human colon cancer cells, and the bioactivity test was a proliferation inhibitory test in KM12 cells, as measured by CCK-8.
The experimental steps are as follows:
1. method for dilution of compound: an appropriate amount of sample was weighed, dissolved in DMSO so that the final concentration of the compound in the dilution was 20mmol/L, and diluted 20-fold with complete medium so that the concentration was 1 mmol/L. The assay was repeated with two-fold dilution, eight concentration gradients, two per well.
2. Cell culture
2.1 preparation of complete medium: DMEM medium was supplemented with 1% double antibody, 10% inactivated fetal bovine serum.
2.2 culture of cells: KM12 cells were inoculated in a medium and incubated at 37 ℃ with 5% CO 2 Culturing in an incubator, and carrying out passage for 2-3 days.
3. Determination of the inhibitory Effect of the samples on the growth of KM12 cells
3.1 KM12 cells were harvested, centrifuged, resuspended, diluted with 5mL DMEM medium and counted. 2000 cells per well, 2X 10 5 The cells were diluted in DMEM medium in the sample addition chamber to a final volume of 8 mL.
3.2 taking 96-well standard plate, marking positive control well, experimental well and blank well.
3.3 Add 80. mu.L cell dilution to each well of positive control and experimental wells, 100. mu.L medium to each well of blank wells, 100. mu.L PBS to each well of well plate periphery to prevent edge effect, incubate 24h in incubator.
3.4 initial concentration of compound 20mM, 10. mu.L was added to 190. mu.L DMEM medium and diluted in 8 gradients in a 96 well V-bottom plate in 2-fold gradients (60. mu.L of diluted sample + 60. mu.L DMEM medium).
3.5 transfer the compounds to the plated cells, 20. mu.L of the test well per well, 20. mu.L of DMEM medium per well of the positive control well, and incubate for 72 h.
3.6 Add 2mL of CCK-8 reagent to the wells and add 10. mu.L of CCK-8 reagent to each well.
3.7 incubate for 1 h.
Absorbance was measured at 450nM with a microplate reader, 630nM as reference, and the final value was 450nM absorbance-630 nM absorbance.
3.9 calculating the inhibition at different concentrations using the inhibition calculation equation, and then calculating the IC using Prism 7 software 50 。
4: the results are shown in FIG. 7 and Table 5.
TABLE 5 human Colon cancer cell IC 50
| |
1 | 2 | 3 |
| ODtop | 59.5100 | 80.0141 | 63.9521 |
| ODBottom | -16.5231 | -3.4661 | -2.4902 |
| IC 50 | 104.5000μmol/L | 46.14μmol/L | 87.3800μmol/L |
| R 2 | 0.9985 | 0.9851 | 0.9964 |
As can be seen, the IC of all-trans teprenone in human colon cancer cells (KM12) 50 Minimum, 46.14 μmol/L; secondly, the mono-cis-teprenone, IC thereof 50 87.38 mu mol/L; again mixed teprenone, its IC 50 The concentration was 104.5. mu. mol/L. It can be concluded that all-trans teprenone plays a major inhibitory role in the proliferation of human colon cancer cells (KM 12).
Example 7 HCT116 human colon cancer cells, bioactivity test proliferation inhibition in HCT116 cells, measured CCK-8.
The experimental steps are as follows:
1. method for dilution of compound: an appropriate amount of sample was weighed, dissolved in DMSO so that the final concentration of the compound in the diluent was 20mmol/L, and diluted 20-fold with complete medium so that the concentration was 1 mmol/L. The assay was repeated with two-fold dilution, eight concentration gradients, two per well.
2. Cell culture
2.1 preparation of complete medium: McCoy's 5a medium was supplemented with 1% double antibody, 10% inactivated fetal bovine serum.
2.2 culture of cells: inoculating HCT116 cells into culture medium, placing at 37 deg.C and 5% CO 2 Culturing in an incubator, and carrying out passage for 2-3 d.
3. Determining the inhibitory Effect of the sample on the growth of HCT116 cells
3.1 HCT116 cells were harvested, centrifuged, resuspended, diluted with 5mL McCoy's 5a medium and counted. 2000 cells per well, 2X 10 5 The cells were diluted in the loading chamber with McCoy's 5a medium to a final volume of 8 mL.
3.2 taking 96-well standard plate, marking positive control well, experimental well and blank well.
3.3 Add 80. mu.L cell dilution to each well of positive control and experimental wells, 100. mu.L medium to each well of blank wells, 100. mu.L PBS to each well of well plate periphery to prevent edge effect, incubate 24h in incubator.
3.4 starting concentration of compound 20mM, 10. mu.L was added to 190. mu.L of McCoy's 5a medium and 8 gradients were diluted in a 2-fold gradient in 96-well V-bottom plates (60. mu.L of diluted sample + 60. mu.L of McCoy's 5a medium).
3.5 transfer of Compounds to the plated cells, 20. mu.L of assay well per well, 20. mu.L of McCoy's 5a medium per well of positive control well, and incubate for 72 h.
3.6 Add 2mL of CCK-8 reagent to the wells and add 10. mu.L of CCK-8 reagent to each well.
Incubate at 3.737 deg.C in carbon dioxide incubator for 1 h.
And 3.8 measuring the absorbance at 450nm by using an enzyme-labeling instrument, taking 630nm as a reference, and obtaining a final value which is 450nm to 630nm absorbance.
3.9 inhibition at various concentrations was calculated using inhibition calculation formula and IC was calculated using Prism 7 software 50 。
4. The results are shown in FIG. 8 and Table 6.
TABLE 6 IC of human colon cancer cells 50
| |
1 | 2 | 3 |
| OD top | 92.1912 | 96.5210 | 84.3962 |
| OD Bottom | -16.5929 | -7.0065 | -10.1950 |
| IC 50 | 26.6500μmol/L | 15.91μmol/L | 23.9700μmol/L |
| R 2 | 0.9952 | 0.9985 | 0.9970 |
It can be seen that, in human colon cancer cells (HCT116), all-transIC of teprenone 50 The minimum is 15.91 mu mol/L; followed by mono-cis-teprenone, its IC 50 23.97 mu mol/L; again mixed teprenone, its IC 50 The concentration was 26.65. mu. mol/L. It can be deduced that the IC of all-trans teprenone versus mixed teprenone 50 More than 1 fold lower, playing a major inhibitory role in human colon cancer cells (HCT 116).
Example 8
293T human embryonic kidney cells, the biological activity test is specifically a test of proliferation inhibition in 293T cells, and the detection method is CCK-8.
The experimental steps are as follows:
1. method of dilution of the compounds: an appropriate amount of sample was weighed, dissolved in DMSO so that the final concentration of the compound in the dilution was 20mmol/L, and diluted 20-fold with complete medium so that the final concentration of the compound in the dilution was 1 mmol/L. The experiment was repeated with two dilutions, eight concentration gradients, two per well.
2. Cell culture
2.1 preparation of complete medium: DMEM medium was supplemented with 1% double antibody, 10% inactivated fetal bovine serum.
2.2 culture of cells: the 293T cells were inoculated into the medium and incubated at 37 ℃ with 5% CO 2 Culturing in an incubator, and carrying out passage for 2-3 d.
3. Determination of the inhibitory Effect of samples on the growth of 293T cells
3.1 collect 293T cells, centrifuge, resuspend, add 5mL DMEM medium to dilute, count cells. 2000 cells per well, 2X 10 5 The cells were diluted in DMEM medium in the sample addition chamber to a final volume of 8 mL.
3.2 taking 96-well standard plate, marking positive control well, experimental well and blank well.
3.3 Add 80. mu.L cell dilution to each well of positive control and experimental wells, 100. mu.L medium to each well of blank wells, 100. mu.L PBS to each well of well plate periphery to prevent edge effect, incubate 24h in incubator.
3.4 starting concentration of compound 20mM, 10. mu.L was added to 190. mu.L DMEM medium and 8 gradients were diluted in a 2-fold gradient in 96-well V-bottom plates (60. mu.L diluted sample + 60. mu.L DMEM medium).
3.5 transfer the compounds to the well plated cells, 20. mu.L of assay well per well, 20. mu.L of DMEM medium per well of positive control well, and incubate for 72 h.
3.6 Add 2mL of CCK-8 reagent to the wells and add 10. mu.L of CCK-8 reagent to each well.
Incubate at 3.737 deg.C for 1h in a carbon dioxide incubator.
And 3.8 measuring the absorbance at 450nm by using an enzyme-labeling instrument, taking 630nm as a reference, and obtaining a final value which is 450nm to 630nm absorbance.
3.9 inhibition at various concentrations was calculated using inhibition calculation formula and IC was calculated using Prism 7 software 50 。
4. The results are shown in FIG. 9 and Table 7.
TABLE 7 human embryonic Kidney cell IC 50
| |
1 | 2 | 3 |
| ODtop | 66.6450 | 81.9598 | 74.4322 |
| ODBottom | -25.6003 | -31.7002 | -31.2459 |
| IC 50 | 57.3200μmol/L | 29.43μmol/L | 63.4000μmol/L |
| R 2 | 0.9743 | 0.9881 | 0.9920 |
As can be seen, the IC of all-trans teprenone in human embryonic kidney cells (293T) 50 Minimum, 29.43 mu mol/L; followed by mixed teprenone, its IC 50 57.32. mu. mol/L; again, the mono-cis-teprenone, IC thereof 50 It was 63.4. mu. mol/L. IC in 293T cells by previous experiments 50 The toxicity is negligible when the concentration is more than 10 mu mol/L, the toxicity of the mixed teprenone is low through clinical verification, and the toxicity of all-trans teprenone can be presumed to be in a reasonable range.
The main reagents and instruments used in the above examples are shown in table 8.
TABLE 8
The present invention has been described in terms of specific examples, which are provided to aid understanding of the invention and are not intended to be limiting. Numerous simple deductions, modifications or substitutions may also be made by those skilled in the art in light of the present teachings.
Claims (10)
1. Application of teprenone in preparing medicine for treating and/or preventing breast cancer, non-small cell lung cancer, lung adenocarcinoma and colon cancer.
2. The use according to claim 1, wherein the teprenone is at least one of all-trans teprenone and single-cis teprenone.
3. The use according to claim 1, wherein the teprenone is all-trans teprenone;
preferably, the molar content of all-trans teprenone in the teprenone is more than or equal to 98%.
4. The use according to claim 1, wherein the teprenone is mono-cis teprenone.
5. The use of claim 1, wherein the teprenone comprises all-trans teprenone, single-cis teprenone;
preferably, the teprenone comprises all-trans teprenone, single-cis teprenone, and the molar ratio of single-cis teprenone: all-trans teprenone (0.6-0.7): 1, preferably (0.61-0.68): 1, more preferably 2: 3.
6. the use of claim 1, wherein the teprenone is for inhibiting proliferation and/or migration of human breast cancer cells.
7. The use of claim 1, wherein the teprenone is for inhibiting proliferation of human non-small cell lung cancer.
8. The use of claim 1, wherein the teprenone is for inhibiting proliferation of human lung adenocarcinoma cells.
9. The use of claim 1, wherein the teprenone is for inhibiting human colon cancer cell proliferation;
and/or, the teprenone is used for inhibiting the proliferation of human rectal cancer cells.
10. The use according to claim 1, wherein the teprenone has low toxicity to normal human cells;
preferably, the IC of the teprenone in human normal cells 50 ＞10μmol/L；
Preferably, the human normal cells include human embryonic kidney cells.
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| CN110882237A (en) * | 2018-09-10 | 2020-03-17 | 厦门信力康生物技术有限公司 | Application of teprenone and derivatives thereof in preparation of medicines for preventing and treating ovarian cancer |
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| HUANBIAO MO ET AL.: "The Potential of Isoprenoids in Adjuvant Cancer Therapy to Reduce Adverse Effects of Statins", 《FRONT. PHARMACOL》, vol. 9, 4 January 2019 (2019-01-04) * |
| POLINA SYSA-SHAH ET AL.,: "Geranylgeranylacetone Blocks Doxorubicin-Induced Cardiac Toxicity and Reduces Cancer Cell Growth and Invasion through RHO Pathway Inhibition", 《MOL CANCER THER》, vol. 13, no. 7, pages 5 * |
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