Application of hydrogen sulfide slow-release donor and cyclodextrin derivative thereof in preparation of medicine for inhibiting migration of triple negative breast cancer cells

Abstract

The invention discloses an application of a hydrogen sulfide slow-release donor and a cyclodextrin derivative thereof in preparation of a medicine for inhibiting migration of triple negative breast cancer cells. According to the method for inhibiting migration of the triple negative breast cancer cells, a donor ADT-OH is slowly released by using hydrogen sulfide. ADT-OH is applied to an in-vitro triple negative breast cancer cell culture system at the concentration of 25-50 [mu] M. According to the method, migration of the triple-negative breast cancer cells is effectively inhibited by using a hydrogen sulfide slow-release donor demethylated anethiol trithione (ADT-OH) and a cyclodextrin modifier (CD-ADT) thereof, so that the migration of the triple-negative breast cancer cells is inhibited, and the discovery of the invention is very likely to provide a clinical drug with a prospect for preventing and treating the triple-negative breast cancer in the future.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to application of a hydrogen sulfide sustained-release donor and a cyclodextrin derivative thereof in preparing a drug for inhibiting migration of triple-negative breast cancer cells. Background Art

[0002] Triple-negative breast cancer (TNBC) is a highly heterogeneous and highly invasive disease with the highest mortality rate among all breast cancer subtypes (Sung et al., 2021, CA Cancer J Clin, 71(3): 209-49). At present, the standard treatment for patients with early triple-negative breast cancer is still conventional chemotherapy, but there is still no unified and effective treatment for patients with metastatic triple-negative breast cancer (André and Zielinski, 2012, Ann Oncol, 23Suppl 6: vi46-vi51). Therefore, it is urgent to develop compounds or new treatment options that effectively inhibit the metastasis of triple-negative breast cancer.

[0003] Hydrogen sulfide (H2S) has become the third largest gas transfer medium after carbon monoxide (CO) and nitric oxide (NO), which can mediate a variety of biological processes, such as cell autophagy, apoptosis, proliferation and metastasis (Wu et al., 2018, Pharmacol Res, 131:120-7; Guo et al., 2012, Oxid Med Cell Longev, 2012:878052). Many studies have found that hydrogen sulfide and its metabolic pathways play an important role in common malignant tumors such as colon cancer, liver cancer, ovarian cancer, and breast cancer (Ngowi et al., 2021, International journal of biological sciences, 17(1):73-88; Wang et al., 2021, Int J Mol Sci, 22(12)). In in vitro and in vivo experiments, large amounts of or long-term low-dose H2S can induce apoptosis of cancer cells (Szabo et al., 2016, Nat Rev Drug Discov, 15(3): 185-203), while having little effect on non-cancerous cells (Cai et al., 2020, Cell Death Dis, 11(1): 33), so H2S donor drugs came into being. At present, many H2S donor drugs have been applied in the field of tumor treatment (Lee et al., 2011, PloS one, 6: e21077; De Cicco et al., 2016, Pharmacol Res, 114: 67-73), such as GYY4137, ATB-346, etc., which have good therapeutic effects on liver cancer, lung cancer, melanoma and other cancers, showing broad application prospects. However, there are few studies on H2S donor drugs in triple-negative breast cancer, especially in inhibiting the metastasis of triple-negative breast cancer. [5-(4-Hydroxyphenyl)-3H-1,2-dithiocyclopentene-3-thione], also known as demethylanisole trisulfide (ADT-OH), is a widely used hydrogen sulfide donor, and previous studies have shown that it has little damage to normal cells and is highly safe. Therefore, the use of ADT-OH to inhibit the metastasis of triple-negative breast cancer may be a potential effective treatment option.

[0004] Cyclodextrins (CD) are a class of cyclic oligosaccharides, which are formed by six or more glucose subunits connected by α-1,4 glycosidic bonds. Therefore, cyclodextrins have a unique ring structure, with a hydrophilic outer surface and a hydrophobic interior. Studies have shown that cyclodextrins are essential for improving the permeability and solubility of anticancer drugs (Yi et al., 2022, Theranostics, 12(6):2560-79). Cyclodextrins can be divided into three categories according to the number of glucose subunits they contain, namely α-cyclodextrin (α-CD), β-cyclodextrin (β-CD) and γ-cyclodextrin (γ-CD). Among them, hydroxypropyl-β-cyclodextrin is a widely used modified cyclodextrin that can effectively improve drug solubility. When molecules of appropriate size interact, non-covalent inclusion complexes are generated; compared with hydrophobic drugs, such inclusion complexes have higher solubility and stability. Previous studies have found that ADT-OH has low permeability and water solubility, and a larger dose must be used for in vivo administration. Therefore, using β-cyclodextrin as a supramolecular drug reservoir to deliver ADT-OH is very likely to effectively enhance the anti-cancer effect of ADT-OH.

[0005] Currently, there is a lack of application of a hydrogen sulfide sustained-release donor ADT-OH and its cyclodextrin derivative CD-ADT in the preparation of drugs for inhibiting the migration of triple-negative breast cancer cells. Summary of the invention

[0006] In order to overcome the deficiencies in the above-mentioned prior art, the purpose of the present invention is to provide a potential treatment scheme for inhibiting the metastasis of triple-negative breast cancer, which inhibits the migration of triple-negative breast cancer cells by using the hydrogen sulfide donor ADT-OH and its modified form hydroxypropyl-β-cyclodextrin-ADT-OH (CD-ADT), thereby developing a triple-negative breast cancer treatment scheme that is simple to operate, has good efficacy, is convenient and safe.

[0007] In order to achieve the above technical objectives, the technical solutions adopted by the present invention are as follows: In the first aspect, the present invention provides a method for inhibiting the migration of triple-negative breast cancer cells. In the second aspect, the present invention provides a cyclodextrin modified CD-ADT of a hydrogen sulfide donor ADT-OH of the present invention. In the third aspect, the present invention provides a method for inhibiting the migration of triple-negative breast cancer cells using a cyclodextrin derivative CD-ADT. In the fourth aspect, the present invention provides a method for applying a compound in an in vivo animal model. In the fifth aspect, the present invention provides a composition. In the sixth aspect, the present invention provides the use of a composition in the preparation of a drug for inhibiting the migration of triple-negative breast cancer cells.

[0008] The method for inhibiting the migration of triple-negative breast cancer cells of the present invention is characterized by using ADT-OH, a hydrogen sulfide slow-release donor.

[0009] Furthermore, the ADT-OH is applied to an in vitro triple-negative breast cancer cell culture system at a concentration of 25 to 50 μM.

[0010] Furthermore, the ADT-OH can significantly reduce the scratch healing area and vertical migration rate of triple-negative breast cancer cells.

[0011] The invention discloses a cyclodextrin modified body CD-ADT of a hydrogen sulfide donor ADT-OH, which is used to enhance the solubility and stability of the hydrogen sulfide sustained-release donor ADT-OH in inhibiting the migration of triple-negative breast cancer cells. The unique cyclic structure of cyclodextrin is used to encapsulate ADT-OH to form a new modified body CD-ADT, and CD-ADT more significantly inhibits the scratch healing of triple-negative breast cancer cells (MDA-MB-231) and the vertical migration rate of two triple-negative breast cancer cells (MDA-MB-231, 4T1-Luci) than ADT-OH alone.

[0012] A method for improving the solubility of hydrogen sulfide donor ADT-OH by cyclodextrin modification and further applying it to tumor treatment, using the unique cyclic structure of cyclodextrin to encapsulate ADT-OH to form a new modified CD-ADT, CD-ADT more significantly inhibits the scratch healing of triple-negative breast cancer cells (MDA-MB-231) and the vertical migration rate of two triple-negative breast cancer cells (MDA-MB-231, 4T1-Luci) than single ADT-OH treatment. The present invention provides a method for inhibiting the in vivo metastasis of triple-negative breast cancer by using hydrogen sulfide donor ADT-OH, which uses different concentrations of ADT-OH to treat the constructed MDA-MB-231 (human triple-negative breast cancer cells) triple-negative breast cancer in situ tumor metastasis model and 4T1-Luci (mouse triple-negative breast cancer cells) triple-negative breast cancer lung metastasis model, respectively, indicating that ADT-OH treatment can significantly inhibit the metastasis of triple-negative breast cancer cells in two animal models.

[0013] 4T1-Luci orthotopic tumor model mice were treated with CD-ADT and different concentrations of ADT-OH, and tumor metastasis was continuously monitored after tumor resection. The modified CD-ADT significantly inhibited in situ recurrence and tumor metastasis after tumor resection surgery compared with ADT-OH treatment alone, and the drug dosage was low.

[0014] Furthermore, the CD-ADT is a non-covalent inclusion complex formed by hydroxypropyl-β-cyclodextrin and ADT-OH.

[0015] The present invention discloses a method for inhibiting the migration of triple-negative breast cancer cells using a cyclodextrin derivative CD-ADT, wherein the cyclodextrin derivative CD-ADT is applied to an in vitro triple-negative breast cancer cell culture system at a concentration of 50 μM. The cyclodextrin derivative CD-ADT shows a better effect of inhibiting the migration of triple-negative breast cancer cells than ADT-OH alone in an in vitro experiment.

[0016] The present invention discloses a method for applying a compound in an in vivo animal model to inhibit the recurrence and metastasis of triple-negative breast cancer, wherein an effective dose of ADT-OH or CD-ADT is administered by injection or oral administration.

[0017] Furthermore, the effective dose is 10 to 30 mg of ADT-OH or CD-ADT per kilogram of body weight.

[0018] A composition of the present invention comprises a hydrogen sulfide sustained-release donor ADT-OH and / or a cyclodextrin derivative CD-ADT thereof.

[0019] Application of the composition of the present invention in preparing a drug for inhibiting the migration of triple-negative breast cancer cells.

[0020] The present invention provides a method for inhibiting in situ recurrence and metastasis of tumor-bearing mice after tumor resection surgery by using ADT-OH cyclodextrin modified body CD-ADT. The method comprises the following steps: using CD-ADT and different concentrations of ADT-OH to treat 4T1-Luci in situ tumor model mice, and continuously monitoring tumor metastasis after tumor resection. The modified CD-ADT significantly inhibits in situ recurrence and tumor metastasis after tumor resection surgery compared with single ADT-OH treatment, and the drug dosage is low.

[0021] Compared with the prior art, the anti-triple-negative breast cancer metastasis drug involved in the present invention has the following innovations and significant improvements: the hydrogen sulfide donor ADT-OH inhibits the in vivo metastasis process of triple-negative breast cancer cells by inhibiting their migration, and the drug dosage is relatively small; the present invention is the first to use cyclodextrin to modify the hydrogen sulfide donor ADT-OH and apply it to tumor treatment, and there has been no related research report so far; the present invention applies the hydrogen sulfide donor ADT-OH and its cyclodextrin modified body CD-ADT to the anti-triple-negative breast cancer metastasis, and demonstrates its use in the preparation of anti-triple-negative breast cancer metastasis and related drugs. This concept is proposed for the first time in the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 A is a representative image of cell scratch healing of the triple-negative breast cancer cell line MDA-MB-231 of the present invention after being treated with 0 μM, 25 μM and 50 μM ADT-OH.

[0024] Figure 1 B is a representative image of cell scratch healing of the triple-negative breast cancer cell line 4T1-Luci of the present invention after being treated with 0 μM, 25 μM and 50 μM ADT-OH.

[0025] Figure 1 C is the present invention Figure 1 A and Figure 1 Statistical results of cell scratch healing in B. (n=3; data are expressed as mean±SD; *p<0.05, **p<0.01)

[0026] Figure 2 This is a graph showing that ADT-OH of the present invention inhibits the vertical migration rate of MDA-MB-231 and 4T1 cells.

[0027] Figure 2 A is a representative image of the Transwell assay of the triple-negative breast cancer cell line MDA-MB-231 of the present invention after treatment with 0 μM, 25 μM and 50 μM ADT-OH.

[0028] Figure 2 B is a representative image of the Transwell assay of the triple-negative breast cancer cell line 4T1-Luci of the present invention after being treated with 0 μM, 25 μM and 50 μM ADT-OH.

[0029] Figure 2 C is the present invention Figure 1 A and Figure 1 Statistical results of cell Transwell assay in B. (n=3; data are expressed as mean±SD; ***p<0.005, ****p<0.001)

[0030] Figure 3 The figure shows that the ADT-OH of the present invention inhibits the metastasis of triple-negative breast cancer cells in the mouse triple-negative breast cancer orthotopic transplant tumor model.

[0031] Figure 3A is a diagram showing liver metastasis in mice after treatment with different concentrations of ADT-OH of the present invention.

[0032] Figure 3 B is the statistical result of the number of liver tumor metastasis nodules in mice after treatment with different concentrations of ADT-OH of the present invention. (n=5; data are expressed as mean±SD; *p<0.05)

[0033] Figure 3 C is a graph showing the results of H&E tissue staining of the liver of each group of mice in the mouse triple-negative breast cancer orthotopic transplantation tumor model of the present invention. (Scale bar = 200 μm; scale bar of the enlarged image = 100 μm)

[0034] Figure 4 The figure shows that the ADT-OH of the present invention inhibits the metastasis of triple-negative breast cancer cells in a mouse triple-negative breast cancer lung metastasis model.

[0035] Figure 4 A is a graph showing the bioluminescence of lung metastatic tumors in mice after treatment with different concentrations of ADT-OH of the present invention.

[0036] Figure 4 B is a graph showing the changes in body weight of mice after treatment with different concentrations of ADT-OH of the present invention (n=5; data are expressed as mean±SD).

[0037] Figure 4 C is a graph showing the survival rate of mice after treatment with different concentrations of ADT-OH of the present invention (n=5; data are expressed as mean±SD; *p<0.05).

[0038] Figure 5 The Fourier transform infrared spectrum and ultraviolet / visible absorption analysis diagram of ADT-OH and its modified body CD-ADT of the present invention.

[0039] Figure 5 A is the Fourier transform infrared spectra of CD, ADT-OH, modified CD-ADT and CD+ADT-OH mixture of the present invention.

[0040] Figure 5 B is the UV-visible spectra of CD, ADT-OH, modified CD-ADT and CD+ADT-OH mixture of the present invention.

[0041] Figure 6 The figure shows that CD-ADT of the present invention inhibits the scratch healing of MDA-MB-231 and 4T1 cells.

[0042] Figure 6A is a representative image of cell scratch healing of the triple-negative breast cancer cell lines MDA-MB-231 and 4T1-Luci of the present invention after treatment with solvent, 50 μM CD-ADT, CD and 50 μM ADT-OH.

[0043] Figure 6 B is the present invention Figure 6 Statistical results of cell scratch healing in A. (n=3; data are expressed as mean±SD; *p<0.05, **p<0.01)

[0044] Figure 7 This is a graph showing that CD-ADT of the present invention inhibits the vertical migration rate of MDA-MB-231 and 4T1 cells.

[0045] Figure 7 A is a representative image of the Transwell assay of the triple-negative breast cancer cell lines MDA-MB-231 and 4T1-Luci of the present invention after treatment with solvent, 50 μM CD-ADT, CD and 50 μM ADT-OH.

[0046] Figure 7 B is the present invention Figure 6 Statistical results of cell Transwell assay in A. (n=3; data are expressed as mean ± SD; *p<0.05, **p<0.01)

[0047] Figure 8 The figure shows that the CD-ADT of the present invention inhibits the metastasis of triple-negative breast cancer cells in the mouse triple-negative breast cancer orthotopic metastasis tumor model.

[0048] Figure 8 A is a graph showing the bioluminescence of tumors in situ and at metastatic sites in mice after treatment with CD-ADT of the present invention and ADT-OH at different concentrations.

[0049] Figure 8 B is a graph showing the effects of CD-ADT of the present invention and different concentrations of ADT-OH on tumor progression in mice with orthotopic tumors. (n=5; data are expressed as mean ± SD)

[0050] Figure 8 C is a graph showing the changes in body weight of mice after treatment with CD-ADT of the present invention and ADT-OH at different concentrations. (n=5; data are expressed as mean ± SD)

[0051] Figure 8 D is a graph showing the survival rate of mice after treatment with CD-ADT of the present invention and ADT-OH at different concentrations. (n=5; data are expressed as mean ± SD; *p<0.05)

[0052] Figure 8E is the present invention Figure 8 A shows the statistics of tumor bioluminescence in situ and metastatic sites in mice after treatment with CD-ADT and different concentrations of ADT-OH. DETAILED DESCRIPTION

[0053] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0054] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0055] The first aspect of the present invention is to provide a method for inhibiting the migration of triple-negative breast cancer cells using ADT-OH, a hydrogen sulfide slow-release donor.

[0056] ADT-OH was applied to the in vitro triple-negative breast cancer cell culture system at concentrations of 25 to 50 μM.

[0057] ADT-OH can significantly reduce the scratch healing area and vertical migration rate of triple-negative breast cancer cells.

[0058] The invention discloses a cyclodextrin modified body CD-ADT of a hydrogen sulfide donor ADT-OH, which is used to enhance the solubility and stability of the hydrogen sulfide sustained-release donor ADT-OH in inhibiting the migration of triple-negative breast cancer cells. The unique cyclic structure of cyclodextrin is used to encapsulate ADT-OH to form a new modified body CD-ADT, and CD-ADT more significantly inhibits the scratch healing of triple-negative breast cancer cells (MDA-MB-231) and the vertical migration rate of two triple-negative breast cancer cells (MDA-MB-231, 4T1-Luci) than ADT-OH alone.

[0059] A method for improving the solubility of hydrogen sulfide donor ADT-OH by cyclodextrin modification and further applying it to tumor treatment, using the unique cyclic structure of cyclodextrin to encapsulate ADT-OH to form a new modified CD-ADT, CD-ADT more significantly inhibits the scratch healing of triple-negative breast cancer cells (MDA-MB-231) and the vertical migration rate of two triple-negative breast cancer cells (MDA-MB-231, 4T1-Luci) than single ADT-OH treatment. The present invention provides a method for inhibiting the in vivo metastasis of triple-negative breast cancer by using hydrogen sulfide donor ADT-OH, which uses different concentrations of ADT-OH to treat the constructed MDA-MB-231 (human triple-negative breast cancer cells) triple-negative breast cancer in situ tumor metastasis model and 4T1-Luci (mouse triple-negative breast cancer cells) triple-negative breast cancer lung metastasis model, respectively, indicating that ADT-OH treatment can significantly inhibit the metastasis of triple-negative breast cancer cells in two animal models.

[0060] 4T1-Luci orthotopic tumor model mice were treated with CD-ADT and different concentrations of ADT-OH, and tumor metastasis was continuously monitored after tumor resection. The modified CD-ADT significantly inhibited in situ recurrence and tumor metastasis after tumor resection surgery compared with ADT-OH treatment alone, and the drug dosage was low.

[0061] CD-ADT is a non-covalent inclusion complex formed by hydroxypropyl-β-cyclodextrin and ADT-OH.

[0062] The second aspect of the present invention is to provide a method for inhibiting the migration of triple-negative breast cancer cells using a cyclodextrin derivative CD-ADT, wherein the cyclodextrin derivative CD-ADT is applied to an in vitro triple-negative breast cancer cell culture system at a concentration of 50 μM. The cyclodextrin derivative CD-ADT shows a better effect of inhibiting the migration of triple-negative breast cancer cells than ADT-OH alone in in vitro experiments.

[0063] The third aspect of the present invention is to provide a method for applying a compound in an in vivo animal model to inhibit the recurrence and metastasis of triple-negative breast cancer, wherein an effective dose of ADT-OH or CD-ADT is administered by injection or oral administration.

[0064] The effective dose is 10 to 30 mg of ADT-OH or CD-ADT per kg body weight.

[0065] The fourth aspect of the present invention is to provide a composition comprising a hydrogen sulfide sustained-release donor ADT-OH and / or its cyclodextrin derivative CD-ADT.

[0066] The fifth aspect of the present invention is to provide a composition for use in preparing a drug for inhibiting the migration of triple-negative breast cancer cells.

[0067] Example 1

[0068] ADT-OH inhibits the scratch healing of MDA-MB-231 and 4T1 cells

[0069] Breast cancer cells MDA-MB-231 and 4T1 in the logarithmic growth phase in a small culture bottle were cultured at 5×10 5 The cells were inoculated into a 6-well plate at a density of 100 cells / well. When the confluence of the monolayer cells reached 90% or more, a 200 μL sterile pipette tip was gently scratched in each well. Then the original culture medium in the well plate was discarded, and after gently washing twice with PBS, it was replaced with a low-serum culture medium containing different concentrations of ADT-OH and co-incubated with the cells. Three replicates were set for each experimental group. At two time points of 0h and 48h, an optical microscope (Carl Zeiss Axioplan2) was used to take pictures and observe the points. Note that the observation position at each time point should be consistent. Finally, the scratch area was measured using ImageJ software and the relative healing area was calculated using the following formula.

[0070]

[0071] Example 2

[0072] ADT-OH inhibits the longitudinal migration of MDA-MB-231 and 4T1 cells

[0073] The culture medium of MDA-MB-231 and 4T1-Luci breast cancer cells in the logarithmic growth phase in the in vitro culture flask was replaced with serum-free DMEM and starved for 12 hours. After starvation, the cells were digested and counted, and the cells were resuspended in DMEM (serum-free) containing different concentrations of ADT-OH to adjust the cell concentration to 5×10 5 / mL. Add 600μL of DMEM complete medium containing 10% FBS to the lower chamber of the Transwell chamber (in the well of the 24-well plate), use tweezers to gently put the upper chamber back into the lower chamber containing complete medium, add 100μL of cell suspension to the upper chamber of the Transwell, continue to culture in the cell culture incubator for 48h, and wait for the cells to penetrate the membrane. After the corresponding time point, take out the upper chamber, discard the culture medium in the upper chamber, and gently wipe off the cells that failed to pass through the membrane with a cotton swab. After rinsing the upper chamber in PBS twice, fix the cells with 4% paraformaldehyde solution at room temperature for 30 minutes, put them in 0.3% crystal violet stain for 30 minutes, and then rinse them with PBS 3 times to wash away the excess stain. Observe under an inverted microscope, take pictures, and count the cell migration.

[0074] Example 3

[0075] ADT-OH inhibits the metastasis of triple-negative breast cancer cells in an orthotopic transplantation model of triple-negative breast cancer in mice

[0076] Select MDA-MB-231 cells that are in good growth state and in the logarithmic growth phase, digest and resuspend them, and adjust the cell concentration to 4×10 7 / mL. The BALB / c nude mice to be operated on were anesthetized with a small animal anesthesia machine, and the outer cortex of the fourth mammary fat pad was cut open to expose the fat pad to the visual field. Next, the cells were mixed with matrix gel at a volume ratio of 1:1 and injected into one side of the fourth pair of mammary fat pads. Each mouse was injected with 100 μL, i.e., 2×10 6 When the tumor grows to 25-40 mm 3 At 14:00, the drug treatment was started every other day. There were three groups of drug administration, with the dosage of 10 mg / kg ADT-OH, 20 mg / kg ADT-OH and 30 mg / kg ADT-OH; the drug solvent (0.5% CMC) was used as a control. At the same time, the tumor size was measured with a digital vernier caliper every 2 days and the weight of the mice was closely monitored. The tumor volume was calculated as follows: Tumor volume = 0.5236 × tumor long axis × tumor short axis 2 Two to three weeks after treatment, mice were euthanized, dissected, weighed, photographed, and desired tissues were fixed in 10% formalin and stained with hematoxylin-eosin (H&E).

[0077] Example 4

[0078] ADT-OH inhibits triple-negative breast cancer cell metastasis in a mouse triple-negative breast cancer lung metastasis model

[0079] Select 4T1-luci cells that are in good growth condition and in the logarithmic growth phase, digest and resuspend them, and adjust the cell concentration to 1.2×10 5 / mL. Take 100 μL and inject it into the mouse through the tail vein, that is, each mouse is injected with 1.2×10 4 cells. On the second day of injection, daily drug administration (100 μL / mouse) was started: 0.5% CMC (drug solvent), 10 mg / kg ADT-OH, 20 mg / kg ADT-OH and 30 mg / kg ADT-OH treatment. One week after administration, tumor metastasis was detected in vivo using a small animal imaging device.

[0080] Example 5

[0081] Fourier transform infrared spectroscopy and UV / visible absorption analysis of ADT-OH and its modified product CD-ADT

[0082] Cyclodextrin (CD), ADT-OH, CD-ADT, and a mixture of CD and ADT-OH were prepared, and KBr pellets were analyzed by Fourier transform infrared spectroscopy (FTIR) on a Bruker TENSOR27 Fourier transform infrared spectrometer (FTIR) to obtain the 4000-500 cm -1 This was followed by UV-visible absorption analysis in a BioTek Synergy H1 microplate reader in the wavelength range of 220 to 999 nm.

[0083] Example 6

[0084] CD-ADT inhibits scratch healing of MDA-MB-231 and 4T1 cells

[0085] Breast cancer cells MDA-MB-231 and 4T1 in the logarithmic growth phase in a small culture bottle were cultured at 5×10 5 The cells were inoculated into a 6-well plate at a density of 100 μL / well. When the confluence of the monolayer cells reached 90% or more, a 200 μL sterile pipette tip was used to gently scratch each well. Then the original culture solution in the well plate was discarded, and after gently washing twice with PBS, it was replaced with a low serum medium containing 0.5% CMC (solvent), 50 μM CD-ADT, CD and 50 μM ADT-OH and the cells were co-incubated. Three replicates were set for each experimental group. Photos were taken and recorded using an optical microscope at two time points of 0h and 48h. Finally, the scratch area was measured using ImageJ software and the relative healing area was calculated using the following formula.

[0086]

[0087] Example 7

[0088] CD-ADT inhibits the vertical migration of MDA-MB-231 and 4T1 cells

[0089] The culture medium of MDA-MB-231 and 4T1-Luci breast cancer cells in the logarithmic growth phase in the in vitro culture flask was replaced with serum-free DMEM and starved for 12 hours. After starvation, the cells were digested and counted, and the cells were resuspended in DMEM (serum-free) containing 0.5% CMC (solvent), 50μM CD-ADT, CD, and 50μM ADT-OH, and the cell concentration was adjusted to 5×10 5 / mL. Add 600μL of DMEM complete medium containing 10% FBS to the lower chamber of the Transwell chamber (in the well of the 24-well plate), use tweezers to gently put the upper chamber back into the lower chamber containing complete medium, add 100μL of cell suspension to the upper chamber of the Transwell, continue to culture in the cell culture incubator for 48h, and wait for the cells to penetrate the membrane. After the corresponding time point, take out the upper chamber, discard the culture medium in the upper chamber, and gently wipe off the cells that failed to pass through the membrane with a cotton swab. After rinsing the upper chamber in PBS twice, fix the cells with 4% paraformaldehyde solution at room temperature for 30 minutes, put them in 0.3% crystal violet stain for 30 minutes, and then rinse them with PBS 3 times to wash away the excess stain. Observe under an inverted microscope, take pictures, and count the cell migration.

[0090] Example 8

[0091] CD-ADT inhibits the metastasis of triple-negative breast cancer cells in a mouse triple-negative breast cancer orthotopic metastasis model

[0092] Select 4T1-luci cells that are in good growth condition and in the logarithmic growth phase, and adjust the cell concentration to 5×10 in PBS after resuspending. 6 / mL. BALB / c nude mice were anesthetized in advance using a small animal anesthesia machine. The cells were injected into the fourth mammary fat pad, with 100 μL per mouse, i.e. 5×10 5 cells. When the tumor was well established, treatment was started every other day: 0.5% CMC (drug solvent), 10 mg / kg CD-ADT, 20 mg / kg ADT-OH and 30 mg / kg ADT-OH treatment. At the same time, the tumor size was measured every 3 days with a digital vernier caliper and the weight of the mice was closely monitored. The tumor volume was calculated as follows: Tumor volume = 0.5236 × tumor long axis × tumor short axis 2 In addition, the mice were imaged before administration to observe the development of the in situ tumor. Animal imaging was continued during administration to observe the progression of the in situ tumor and the occurrence of tumor metastasis. On the 18th day after tumor inoculation, the tumor volume was basically 800mm 3 At this time, the in situ tumor resection surgery is started, and the tissue around the tumor is removed as cleanly as possible, and then the spontaneous metastasis of the tumor is observed (caused by the shedding of the original tumor cells). After the tumor is removed, the animal imaging is immediately started after the wound heals well to observe whether the tumor is completely removed. After that, the animal imaging is continued and the spontaneous metastasis in the body is detected in vivo. After the tumor is removed, the treatment is continued for about three weeks, during which the drug is continuously administered and the tumor metastasis is observed.

[0093] The present invention uses a cell scratch healing assay and a Transwell assay to detect the effect of hydrogen sulfide donor ADT-OH treatment on the migration of MDA-MB-231 and 4T1-Luci cells. The results of the cell scratch assay showed that the healing area of ​​the two triple-negative breast cancer cells was significantly reduced after ADT-OH treatment ( Figure 1 ); The results of the Transwell experiment also showed that the number of cells that penetrated the membrane of the two triple-negative breast cancer cells was significantly reduced after ADT-OH treatment ( Figure 2 ), and the inhibitory effect of ADT-OH was dose-dependent. Therefore, ADT-OH significantly inhibited the planar and spatial migration ability of triple-negative breast cancer cells.

[0094] Next, in order to verify the anti-metastatic properties of ADT-OH in triple-negative breast cancer, a mouse orthotopic tumor metastasis model ( Figure 3 ), Tumor lung metastasis model ( Figure 4 ) detected the inhibitory effect of ADT-OH on triple-negative breast cancer metastasis in vivo. By counting the number of mouse metastatic tumor nodules, tissue H&E staining, body weight and survival analysis, and tumor bioluminescence analysis, it was found that ADT-OH significantly inhibited the metastasis of triple-negative breast cancer cells in two mouse metastasis models, and showed significant anti-tumor cell metastasis effects at a relatively low therapeutic dose (10 mg / kg).

[0095] Then, in order to further improve the therapeutic effect of ADT-OH, the modified ADT-OH CD-ADT ( Figure 4 ). The effects of CD-ADT and ADT-OH at the same dose on the migration of MDA-MB-231 and 4T1-Luci cells were detected by cell scratch healing and Transwell assay. The cell scratch assay showed that CD-ADT significantly inhibited the scratch healing of MDA-MB-231 cells compared with ADT-OH treatment alone ( Figure 5 ); Transwell results also showed that CD-ADT could more significantly inhibit the vertical migration of two types of triple-negative breast cancer cells than ADT-OH alone. This shows that CD-ADT can more effectively inhibit the migration of triple-negative breast cancer cells.

[0096] Next, 4T1-Luci orthotopic tumor model mice were used and tumor metastasis was continuously monitored after tumor resection. Through the analysis and recording of the body weight and survival period of the model mice and the statistical analysis of tumor bioluminescence, it was found that CD-ADT (10 mg / kg) and different concentrations of ADT-OH treatment significantly inhibited tumor recurrence and metastasis after tumor resection surgery. In addition, the survival period of CD-ADT-treated mice was significantly increased, and the anti-tumor metastasis effect was better than that of ADT-OH treatment alone, and the drug dosage was also less.

[0097] Therefore, the present invention provides a potential treatment scheme for inhibiting the metastasis of triple-negative breast cancer cells by using hydrogen sulfide donor ADT-OH and its modified body CD-ADT. The scheme inhibits the migration of triple-negative breast cancer cells by using hydrogen sulfide donor ADT-OH and its modified body hydroxypropyl-β-cyclodextrin-ADT-OH, and has simple operation and good efficacy. Its characteristic is that the present invention first proposes the application of ADT-OH and its modified body CD-ADT in the study of inhibiting the metastasis of triple-negative breast cancer, which provides a new idea and new direction for the development of anti-triple-negative breast cancer metastasis drugs and drug applications.

[0098] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and the scope of protection of the present invention is defined by the attached claims, description and their equivalents.

Claims

1. A method for inhibiting the migration of triple-negative breast cancer cells, characterized in that ADT-OH, a hydrogen sulfide slow-release donor, was used.

2. The method for inhibiting the migration of triple-negative breast cancer cells according to claim 1, characterized in that: The ADT-OH is applied to an in vitro triple-negative breast cancer cell culture system at a concentration of 25 to 50 μM.

3. The method for inhibiting the migration of triple-negative breast cancer cells according to claim 1 or 2, characterized in that: The ADT-OH can significantly reduce the scratch healing area and vertical migration rate of triple-negative breast cancer cells.

4. A cyclodextrin modified product CD-ADT of hydrogen sulfide donor ADT-OH, characterized in that: Used to enhance the solubility and stability of the hydrogen sulfide sustained-release donor ADT-OH described in claim 1 in inhibiting the migration of triple-negative breast cancer cells.

5. The cyclodextrin derivative CD-ADT according to claim 4, characterized in that: The CD-ADT is a non-covalent inclusion complex formed by hydroxypropyl-β-cyclodextrin and ADT-OH.

6. A method for inhibiting the migration of triple-negative breast cancer cells using the cyclodextrin derivative CD-ADT according to claim 4 or 5, characterized in that: The cyclodextrin derivative CD-ADT is applied to an in vitro triple-negative breast cancer cell culture system at a concentration of 50 μM.

7. A method of using the compound of claim 1 or 4 in an in vivo animal model to inhibit the recurrence and metastasis of triple-negative breast cancer, characterized in that An effective dose of ADT-OH or CD-ADT is administered by injection or oral administration.

8. The method according to claim 7, characterized in that The effective dose is 10 to 30 mg of ADT-OH or CD-ADT per kg of body weight.

9. A composition comprising the hydrogen sulfide sustained-release donor ADT-OH and / or its cyclodextrin derivative CD-ADT.

10. Use of the composition according to claim 9 in the preparation of a drug for inhibiting the migration of triple-negative breast cancer cells.

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