Pharmaceutical composition for inhibiting proliferation and migration of triple negative breast cancer cells and application of pharmaceutical composition

Abstract

The invention provides a pharmaceutical composition for inhibiting proliferation and migration of triple negative breast cancer cells and application thereof. The pharmaceutical composition comprises preparation of the pharmaceutical composition and cell culture. The preparation method of the pharmaceutical composition comprises the following steps: accurately weighing trametes robiniophila clear paste, dissolving the trametes robiniophila clear paste in an RPMI1640 or DMEM complete medium until the final concentration is 100mg / ml, filtering and sterilizing by using a 0.22 mu m filter, and storing in a refrigerator at-20 DEG C for later use; dissolving colchicine in DMSO (dimethylsulfoxide) to prepare 50 [mu] M mother liquor, and storing for later use; when in use, the two medicines are diluted into proper concentrations according to the proportion; the step of cell culture comprises the substeps that mouse breast cancer cells 4T1 and human breast cancer cells MDA-MB-231 are cultured, and the 4T1 cells and the MDA-MB-231 cells are cultured in an RPMI1640 culture medium and a DMEM culture medium respectively; the pharmaceutical composition can inhibit cell viability and cause cell morphological change in 4T1 cells and MDA-MB-231 cells, the pharmaceutical composition can significantly inhibit the viability of the 4T1 cells and the MDA-MB-231 cells in a dose-dependent manner, most of the cells become larger, cytotoxicity is more obvious after 48 hours and 72 hours, the viability of the 4T1 cells is reduced by 39%, the activity of the MDA-MB-231 cells is reduced by 39%, the activity of the MDA-MB-231 cells is reduced by 39%, and the activity of the MDA-MB-231 cells is reduced by 39%. And the activity of the MDA-MB-231 cells is reduced by 78%.

Description

Technical Field

[0001] The present invention belongs to the field of medical biotechnology, and particularly relates to a pharmaceutical composition for inhibiting proliferation and migration of triple-negative breast cancer cells and an application thereof. Background Art

[0002] There are many conventional treatments for breast cancer, including surgery, radiotherapy, chemotherapy, hormone therapy, and other adjuvant therapies. However, these therapies cannot completely prevent cancer recurrence and metastasis, so cancer patients are in great need of new drugs and therapies. In adjuvant therapy, traditional Chinese medicine (TCM) is increasingly widely used because of its unique effect in killing cancer cells. In Asia, Chinese medicine has a history of three thousand years, and 62% of anti-infective and anti-cancer drugs that can be commercially applied or in later development are drugs derived from nature. Chinese medicine contains a variety of complex compounds, such as alkaloids, steroids, and proteoglycans, which can produce unique anti-cancer effects at safe doses.

[0003] Sophora auricula is a kind of medicinal fungus in China, and its Chinese medicine name is Sophora auricula. The anti-tumor effect of Sophora auricula extract shows multiple biological activities, such as cell apoptosis, drug resistance reversal, anti-metastasis and immune system activation. Considering the characteristics of Sophora auricula, a pharmaceutical composition for inhibiting the proliferation and migration of triple-negative breast cancer cells and its application are proposed. Summary of the invention

[0004] The present invention provides a pharmaceutical composition for inhibiting the proliferation and migration of triple-negative breast cancer cells and application thereof, which solves the problems in the prior art.

[0005] The technical solution of the present invention is achieved as follows: a pharmaceutical composition for inhibiting the proliferation and migration of triple-negative breast cancer cells and its application, including the preparation of the pharmaceutical composition and cell culture;

[0006] The preparation of the pharmaceutical composition includes Sophora auricula paste and colchicine. Sophora auricula paste is accurately weighed and dissolved in RPMI1640 or DMEM complete medium to a final concentration of 100 mg / ml, filtered and sterilized with a 0.22 μm filter, and stored in a -20°C refrigerator for standby use; colchicine is dissolved in DMSO to prepare a 50 uM stock solution for storage and standby use, and diluted to 0.1 uM during work; and the two drugs are diluted to a suitable concentration according to the ratio when used;

[0007] Cell culture includes mouse breast cancer cells 4T1 and human breast cancer cells MDA-MB-231. 4T1 cells and MDA-MB-231 cells are cultured in RPMI1640 and DMEM culture media, respectively.

[0008] As a preferred embodiment, the RPMI1640 or DMEM culture medium contains 10% FBS, 1% penicillin and streptomycin, and the RPMI1640 and DMEM culture medium are used to culture 4T1 cells and MDA-MB-231 cells overnight, respectively.

[0009] Furthermore, the cell viability was measured to ensure the accuracy of the data.

[0010] As a preferred embodiment, the RPMI1640 or DMEM culture medium contains a 96-well culture plate.

[0011] As a preferred embodiment, the pharmaceutical composition replaces RPMI1640 and DMEM culture medium, and is cultured alone for 24, 48 or 72 hours, and 20 μL of MTT is added to a 96-well culture plate, and the cells are cultured at 37°C for 4 hours. After 4 hours, the supernatant is aspirated, 100 μL of DMSO is added to the 96 wells, and the plates are shaken for 10 minutes, and then the absorbance value is read by a microplate reader.

[0012] As a preferred embodiment, the concentrations of the pharmaceutical composition are 0, 2, 4, 8, and 16 mg / mL of Sophora japonica fungus and 0.1 uM of colchicine.

[0013] As a preferred embodiment, the pharmaceutical composition at a concentration of 4 mg / mL is used to culture 4T1 cells and MDA-MB-231 cells for 24 and 48 hours, and the morphology of the treated cells and the negative control cells is observed under an optical microscope.

[0014] As a preferred embodiment, 4T1 cells and MDA-MB-231 cells were cultured with the pharmaceutical composition at a concentration of 4 mg / mL for 24 and 48 hours, and the cell scratching state was observed under an optical microscope.

[0015] Furthermore, the pharmaceutical composition inhibits the viability of MDA-MB-231 cells. MDA-MB-231 cells were treated with the pharmaceutical composition for 0, 24, and 48 hours. The pharmaceutical composition significantly inhibited the cell viability of the two cell lines in a time- and dose-dependent manner.

[0016] After adopting the above technical scheme, the beneficial effect of the present invention is: in 4T1 cells and MDA-MB-231 cells, after the cells are treated with the drug composition for 24, 48 and 72 hours in a dose-dependent manner, the MTT test is used to determine the cell viability, wherein the drug composition significantly inhibits 4T1 cells and MDA-MB-231 cells in a time- and dose-dependent manner, and the cell viability decreases sharply at 8 mg / mL, and the cytotoxicity of 4T1 cells and MDA-MB-231 cells is more obvious at 48 and 72 hours, the viability of 4T1 cells decreases by 39%, and the viability of MDA-MB-231 cells decreases by 78%.

[0017] Cells exposed to the drug composition have reduced motility.

[0018] The 4T1 cells and MDA-MB-231 cells are in a culture medium containing a drug composition, and the drug composition can block the cell cycle and inhibit cell proliferation. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0020] Figure 1 This is a schematic diagram of the morphological changes of MDA-MB-231 and 4T1 cells of the present invention;

[0021] Figure 2 Schematic diagram of the scratch changes of MDA-MB-231 and 4T1 cells of the present invention; DETAILED DESCRIPTION

[0022] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] Example 1

[0024] A pharmaceutical composition for inhibiting the proliferation and migration of triple-negative breast cancer cells and its application, including preparation of the pharmaceutical composition and cell culture;

[0025] The preparation of the pharmaceutical composition includes Sophora auricula paste and colchicine. Sophora auricula paste is accurately weighed and dissolved in RPMI1640 or DMEM complete medium to a final concentration of 100 mg / ml, filtered and sterilized with a 0.22 μm filter, and stored in a -20°C refrigerator for standby use; colchicine is dissolved in DMSO to prepare a 50 uM stock solution for storage and standby use, and diluted to 0.1 uM during work; and the two drugs are diluted to a suitable concentration according to the ratio when used;

[0026] Cell culture includes mouse breast cancer cells 4T1 and human breast cancer cells MDA-MB-231. 4T1 cells and MDA-MB-231 cells are cultured in RPMI1640 and DMEM culture media, respectively.

[0027] RPMI1640 or DMEM medium contained 10% FBS, 1% penicillin and streptomycin. RPMI1640 and DMEM medium were used to culture 4T1 cells and MDA-MB-231 cells overnight, respectively.

[0028] Furthermore, the cell viability was measured to ensure the accuracy of the data.

[0029] 96-well culture plates were contained in RPMI1640 or DMEM medium.

[0030] The drug composition replaced RPMI1640 or DMEM culture medium and was cultured alone for 24, 48 or 72 hours, and 20 μL of MTT was added to a 96-well culture plate, and the cells were cultured at 37°C for 4 hours. After 4 hours, the supernatant was aspirated, 100 μL of DMSO was added to the 96 wells, shaken for 10 minutes, and then the absorbance value was read by a microplate reader.

[0031] The concentrations of the drug composition are 0, 2, 4, 8, 16 mg / mL of Sophora japonica fungus and 0.1 uM of colchicine.

[0032] The pharmaceutical composition at a concentration of 4 mg / mL was used to culture 4T1 cells and MDA-MB-231 cells for 24 and 48 hours, and the morphology of the treated cells and the negative control cells was observed under an optical microscope.

[0033] The pharmaceutical composition containing a concentration of 4 mg / mL was used to culture 4T1 cells and MDA-MB-231 cells for 24 and 48 hours, and the cell scratch state was observed under an optical microscope.

[0034] Furthermore, the pharmaceutical composition inhibits the viability of MDA-MB-231 cells. MDA-MB-231 cells were treated with the pharmaceutical composition for 0, 24, and 48 hours. The pharmaceutical composition significantly inhibited the cell viability of both cell lines in a time- and dose-dependent manner.

[0035] like Figure 1-2 As shown, 0, 24, and 48 hours are the cell change states of the drug composition 4T1 cells and MDA-MB-231 cells and the scratch change states. The drug composition strongly inhibits the cell motility, and the reduction in cell number indicates that the inhibitory effect of the drug composition is significant.

[0036] The present invention provides a pharmaceutical composition for inhibiting the proliferation and migration of triple-negative breast cancer cells and its application. The pharmaceutical composition can strongly inhibit the cell motility, inhibit the proliferation of triple-negative breast cancer cells, and promote the apoptosis of triple-negative breast cancer cells, which brings new ideas for the treatment of triple-negative breast cancer patients and opens up a new application field of the pharmaceutical composition. The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. A pharmaceutical composition for inhibiting the proliferation and migration of triple-negative breast cancer cells and its application, characterized in that: Including preparation of pharmaceutical compositions and cell culture; The preparation of the pharmaceutical composition includes Sophora auricula paste and colchicine. Sophora auricula paste is accurately weighed and dissolved in RPMI1640 or DMEM complete medium to a final concentration of 100 mg / ml, filtered and sterilized with a 0.22 μm filter, and stored in a -20°C refrigerator for standby use; colchicine is dissolved in DMSO to prepare a 50 uM stock solution for storage and standby use, and diluted to 0.1 uM during work; and the two drugs are diluted to a suitable concentration according to the ratio when used; Cell culture includes mouse breast cancer cells 4T1 and human breast cancer cells MDA-MB-231. 4T1 cells and MDA-MB-231 cells are cultured in RPMI1640 and DMEM culture media, respectively.

2. A pharmaceutical composition for inhibiting the proliferation and migration of triple-negative breast cancer cells according to claim 1 and its use, characterized in that: The RPMI1640 or DMEM culture medium contains 10% FBS, 1% penicillin and streptomycin. The RPMI1640 and DMEM culture medium are used to culture 4T1 cells and MDA-MB-231 cells overnight, respectively.

3. A pharmaceutical composition for inhibiting the proliferation and migration of triple-negative breast cancer cells and its use according to claim 2, characterized in that: The RPMI1640 or DMEM culture medium contains a 96-well culture plate.

4. A pharmaceutical composition for inhibiting the proliferation and migration of triple-negative breast cancer cells and its use according to claim 3, characterized in that: The drug composition replaces RPMI1640 and DMEM culture medium, and is cultured alone for 24, 48 or 72 hours, and 20 μL of MTT is added to a 96-well culture plate, and the cells are cultured at 37° C. for 4 hours. After 4 hours, the supernatant is aspirated, 100 μL of DMSO is added to the 96 wells, and the plates are shaken for 10 minutes, and then the absorbance value is read by an enzyme plate reader.

5. A pharmaceutical composition for inhibiting the proliferation and migration of triple-negative breast cancer cells and its use according to claim 1, characterized in that: The concentrations of the pharmaceutical composition are 0, 2, 4, 8, and 16 mg / mL, respectively, of Sophora japonica fungus combined with 0.1 uM colchicine.

6. A pharmaceutical composition for inhibiting the proliferation and migration of triple-negative breast cancer cells and its use according to claim 2, characterized in that: The pharmaceutical composition at a concentration of 4 mg / mL was used to culture 4T1 cells and MDA-MB-231 cells for 24 and 48 hours, and the morphology of the treated cells and the negative control cells was observed under an optical microscope.

7. A pharmaceutical composition for inhibiting the proliferation and migration of triple-negative breast cancer cells and its use according to claim 1, characterized in that: The pharmaceutical composition containing a concentration of 4 mg / mL was used to culture 4T1 cells and MDA-MB-231 cells for 24 and 48 hours, and the cell scratch state was observed under an optical microscope.

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