CN112870193B

CN112870193B - Application of melatonin in preparation of medicine for treating HER2 positive breast cancer resistant to targeted medicine

Info

Publication number: CN112870193B
Application number: CN202110265982.7A
Authority: CN
Inventors: 程海凌; 刘丕旭; 桑晓琳; 刘尊东
Original assignee: Second Hospital of Dalian Medical University
Current assignee: Second Hospital of Dalian Medical University
Priority date: 2021-03-11
Filing date: 2021-03-11
Publication date: 2022-07-08
Anticipated expiration: 2041-03-11
Also published as: CN112870193A

Abstract

The invention discloses an application of melatonin in preparing a medicament for treating HER2 positive breast cancer with drug resistance to a targeted medicament. The research of the invention shows that the melatonin can play a role in treating HER2 positive breast cancer by regulating the stability of HER2 protein; in addition, the research proves that the melatonin can still enhance the treatment effect of HER2 small molecule targeted drugs on HER2 positive breast cancer resistant to HER2 small molecule targeted drugs, and the melatonin can be used for preventing recurrence and metastasis of HER2 positive breast cancer. The research result of the invention provides a new treatment idea for clinically preventing the recurrence and metastasis of HER2 positive breast cancer.

Description

Application of melatonin in preparation of medicine for treating HER2 positive breast cancer resistant to targeted medicine

Technical Field

The invention belongs to the field of biomedicine, and relates to application of melatonin in preparation of a medicine for treating HER2 positive breast cancer with drug resistance to a targeted medicine.

Background content

In recent years, with the wide application of the HER2 monoclonal antibody and the small molecule targeted inhibitor in clinical treatment, the prognosis of a HER2 positive breast cancer patient is remarkably improved, but primary or secondary drug resistance is still an important challenge facing the clinical treatment of HER2 positive breast cancer.

Lapatinib (Lapatinib) is an oral dual tyrosine kinase inhibitor aiming at HER2/neu and EGFR, can be reversibly combined with an intracellular catalytic domain of a receptor, inhibits the activation of the receptor, blocks the signal transduction of downstream PI3K/AKT and Ras/Raf/ERK/MAPK pathways, and can effectively inhibit the growth and survival of herceptin-resistant HER2 positive breast cancer cells. Lapatinib binds reversibly to the ATP binding site of the tyrosine kinase domain through hydrogen bonds, inhibits autophosphorylation and activation of tyrosine kinase, and activation of PI3K/AKT and MAPK downstream of EGFR/HER2, thereby inhibiting proliferation of cells and inducing apoptosis of tumor cells. Although lapatinib has a good treatment effect on HER2 positive breast cancer, primary or secondary drug resistance of lapatinib exists in some patients, so that the curative effect of lapatinib in breast cancer, particularly advanced breast cancer, is limited, and the lapatinib is a difficult problem in clinic. There are several mechanisms currently responsible for lapatinib resistance, such as: mTORC1 activation independent of the PI3K pathway, ERR α -mediated metabolic adaptation, overexpression of β 1 integrin, autocrine mitogenic signaling, and the like. Therefore, the search of a coping strategy after HER2 targeted drug resistance has important clinical guidance significance.

Neratinib (Neratinib) is a new generation of small molecule inhibitors of irreversible receptor tyrosine kinases (HER1/EGFR, HER2 and HER4) that have recently been approved for the treatment of HER2 positive breast cancer. There are studies showing that: (1) the mutation at the T798I site of HER2 may be associated with acquired resistance to Neratinib, and the T798I mutation is located in the intracellular enzymatically active region of HER2, preventing Neratinib from binding to HER2, resulting in resistance to Neratinib. (2) In another study for screening Neratinib drug-resistant targets by using a small interfering RNA lentivirus library targeting whole genome, it is found that expression down-regulation of a series of genes such as oncogenes RAB33A, RAB6A and BCL2L14, nuclear transcription factors FOXP4, TFEC and ZNF, cell ion transport-related proteins CLIC3, TRAPPC2P1 and P2RX2, cell ubiquitin-related proteins UBL5 and cell cycle-related proteins CCNF in HER2 positive breast cancer cell lines SKBR3 can cause Neratinib drug resistance. (3) Neuregulin U (NmU) can stabilize the expression of HER2 protein by USP27, therefore high expression of NmU stably expresses HER2 on cell membrane, resulting in the development of drug resistance of tumors against HER2 targeted inhibitors trastuzumab, Lapatinib and Neratinib. The researches show that primary or secondary drug resistance of Neratinib is unavoidable in clinical treatment application, so that an effective combined drug administration scheme is searched, and the enhancement of the curative effect of Neratinib is a problem to be solved urgently in clinical treatment of HER2 positive breast cancer.

Melatonin (Melatonin), a small lipophilic indoleamine produced by pineal gland, has been shown to inhibit tumor cells in many tumor cells. Melatonin (Melatonin) is a naturally occurring hormone with low cytotoxicity and good safety. In addition to enhancing efficacy, melatonin has also been shown in various animal models and clinical studies to reduce adverse effects or toxicity caused by chemotherapy or radiation. There are studies reporting that melatonin can inhibit the expression of proteins such as HER2, MAPK and mTOR in rat ovarian cancer, but there is no specific mechanism reported. Whether melatonin affects the expression of HER2 protein in human HER2 positive breast cancer is not reported, and whether the melatonin is combined with HER2 targeted inhibitor Neratinib to treat HER2 positive breast cancer is not reported.

One of the keys to achieving an effective HER 2-targeted therapy is the complete blockade of HER2 protein and its downstream signaling pathways. Although treatment with HER2 has been highly successful, in a significant proportion of patients with HER2 positive breast cancer, disease will recur. How to improve the therapeutic efficacy of existing HER 2-targeted drugs remains an unmet clinical need.

Disclosure of Invention

The research of the invention proves that the melatonin can play a role in treating HER2 positive breast cancer by adjusting the stability of HER2 protein; in addition, the research proves that the melatonin can still enhance the treatment effect of HER2 small molecule targeted drugs on HER2 positive breast cancer resistant to HER2 small molecule targeted drugs, and the melatonin can be used for preventing recurrence and metastasis of HER2 positive breast cancer.

According to one aspect of the invention, the invention provides the use of melatonin in the manufacture of a medicament for the treatment of HER2 positive breast cancer.

Further, the HER2 positive breast cancer includes HER2 positive breast cancer that is resistant or intolerant to HER2 small molecule targeted drugs.

Still further, the HER2 small molecule targeted drug includes a tyrosine kinase inhibitor.

Still further, the tyrosine kinase inhibitor comprises lapatinib, lenatinib, tucanitinib, pyrroltinib, afatinib, pozzatinib, dacomitinib, perotinib (pirotinib).

In a specific embodiment of the invention, the tyrosine kinase inhibitor is lapatinib or lenatinib.

According to another aspect of the invention, there is provided the use of melatonin in the manufacture of a medicament for preventing recurrence or metastasis of HER2 positive breast cancer.

According to a further aspect of the invention, the invention provides the use of melatonin for the preparation of a medicament for reducing the stability of the HER2 protein or for promoting the degradation of the HER2 protein.

According to a further aspect of the invention, the invention provides the use of melatonin in the manufacture of a medicament for promoting degradation of HER2 protein.

According to yet another aspect of the invention, the invention provides the use of melatonin in the preparation of a medicament for enhancing the killing effect of a small molecule targeted medicament HER2 on HER2 positive breast cancer cells.

Preferably, the drug comprises melatonin, HER2 small molecule targeted drugs; preferably, HER2 positive breast cancer includes HER2 positive breast cancer that is resistant or intolerant to HER2 small molecule targeted drugs.

According to another aspect of the invention, the invention provides an application of melatonin in preparing a medicament for enhancing the treatment effect of a HER2 small molecule targeted medicament on HER2 positive breast cancer.

Preferably, the drug comprises melatonin, HER2 small molecule targeted drugs; preferably, the HER2 positive breast cancer comprises HER2 positive breast cancer that is resistant or intolerant to HER2 small molecule targeted drugs.

According to another aspect of the invention, the invention provides an application of melatonin and a HER2 small molecule targeted drug in preparing a drug for enhancing the killing effect of the HER2 small molecule targeted drug on HER2 positive breast cancer or the treatment effect on HER2 positive breast cancer; preferably, HER2 positive breast cancer includes HER2 positive breast cancer that is resistant or intolerant to HER2 small molecule targeted drugs.

Further, the HER2 small molecule targeted drug includes a tyrosine kinase inhibitor.

Further, tyrosine kinase inhibitors include lapatinib, neratinib, cartinib, pirtinib, afatinib, pozzertib, dacomitinib, perotinib (pirotinib).

According to yet another aspect of the invention, the invention provides a pharmaceutical composition for the treatment of HER2 positive breast cancer comprising melatonin and a HER2 small molecule targeted drug. Preferably, the HER2 positive breast cancer comprises HER2 positive breast cancer that is resistant or intolerant to HER2 small molecule targeted drugs.

Further, the tyrosine kinase inhibitor comprises lapatinib, neratinib, cartinib, pirtinib, afatinib, pozzertib, dacomitinib, perotinib (pirotinib).

According to yet another aspect of the invention, the invention provides the use of melatonin and a HER2 small molecule targeted drug in the preparation of a medicament for the treatment of HER2 positive breast cancer.

Preferably, HER2 positive breast cancer includes HER2 positive breast cancer that is resistant or intolerant to HER2 small molecule targeted drugs.

According to yet another aspect of the invention, there is provided a method of preventing relapse or metastasis in a HER2 positive breast cancer patient, the method comprising administering melatonin to a HER2 positive breast cancer patient.

Preferably, the method comprises administering melatonin and a tyrosine kinase inhibitor to a HER2 positive breast cancer patient. The tyrosine kinase inhibitor may be administered first, followed by melatonin; the tyrosine kinase inhibitor and melatonin may also be administered simultaneously.

According to yet another aspect of the invention, there is provided a method of promoting apoptosis in HER2 positive breast cancer cells resistant to a small molecule targeted drug of HER2 comprising administering melatonin.

Furthermore, the dosage form of the medicine is any pharmaceutically acceptable dosage form. Including but not limited to tablets (including dispersible tablets, enteric-coated tablets, chewable tablets, orally disintegrating tablets, effervescent tablets, etc.), hard capsules (including enteric-coated capsules), soft capsules, granules, pills, micro-pills, dropping pills, dry suspensions, oral solutions, dry syrups, powders, oral suspensions, oral quick-release or slow-release or controlled-release dosage forms, injections (including sterile powder injections for injection and freeze-dried powder injections), aqueous solution injections, ointments, gels, emulsions, patches, suppositories, gels, and the like.

The medicament of the invention can be applied by oral route, and can also be administrated by intravenous, intramuscular, intradermal or subcutaneous injection route.

The medicine of the invention can be used alone or combined with other medicines for treating breast cancer. Other drugs for treating breast cancer include those disclosed in the prior art. The drug of the present invention and other drugs may be administered simultaneously, or the drug of the present invention may be administered first followed by the other drugs, or the other drugs may be administered first followed by the drug of the present invention.

The medicaments of the present invention may also include pharmaceutically acceptable ingredients including, but not limited to, pharmaceutically acceptable carriers, diluents, excipients, adjuvants, buffers, pH adjusting agents, preservatives, antioxidants, bacteriostats, stabilizers, suspending agents, solubilizers, surfactants (e.g., wetting agents), coloring agents, and isotonicizing solutes (i.e., which render the formulation isotonic with the blood or other relevant bodily fluids of the subject patient). Suitable carriers, diluents, excipients and the like can be found in standard pharmaceutical books. See, e.g., the handbook of Pharmaceutical Additives (handbook), second edition (editors m.ash and i.ash), 2001(SynapseInformation Resources, inc., endiott, New York, USA); remington's pharmaceutical Science, 18 th edition, Mack Publishing Company, Easton, Pa., 1990; and the Handbook of pharmaceutical Excipients (Handbook of pharmaceutical Excipients), second edition, 1994.

The term "pharmaceutically acceptable" as used herein refers to compounds, ingredients, materials, compositions, dosage forms, and the like, which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of patients without undue undesirable toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

In this specification and claims, the words "comprise", "comprising" and "contain" mean "including but not limited to", and are not intended to exclude other moieties, additives, components or steps.

The invention has the advantages and beneficial effects that:

the research of the invention proves that the melatonin can effectively reduce the expression level of HER2 protein, thereby obviously inhibiting the in vitro cell line level of HER2 positive breast cancer cells and the growth of tumors in mice. The research suggests that melatonin is used as a treatment drug for effectively resisting HER2 positive breast cancer, and provides a new idea for a combined treatment strategy of double-blocking HER2 based on a HER2 small-molecule targeted treatment drug (such as Neratinib and Lapatinib) which is clinically approved and applied.

The research of the invention proves that the melatonin and the small molecule HER2 targeted drug are combined for use, so that the strong anti-tumor activity is generated under the condition that the weight of a mouse is not obviously changed, and the potential of the melatonin as an adjuvant for combined treatment of HER2 positive breast cancer is supported.

The study of the invention proves that Melatonin (Melatonin) can effectively enhance the sensitivity of drug-resistant breast cancer cells to HER2 small-molecule targeted therapeutic drugs (Neratinib and Lapatinib), and the study suggests that the combined drug strategy is expected to relieve or even overcome the phenomenon of clinical HER2 positive breast cancer drug-resistant relapse.

Drawings

FIG. 1 shows a flow cytometry analysis of the effect of different concentrations of melatonin on the death of HER2 positive breast cancer cells HCC1954, MDA-MB-361 and MCF7/HER2, wherein A: flow cytometry detection cell death result graph, B: a quantitative map;

FIG. 2 shows the effect of different concentrations of melatonin on total HER2 protein expression in HER2 positive breast cancer cells HCC1954, MDA-MB-361 and MCF7/HER2, respectively, using Western immunoblotting;

FIG. 3 shows a graph of flow cytometry to determine the effect of melatonin at various concentrations on HER2 positive breast cancer cells HCC1954, MDA-MB-361 and MCF7/HER2 cell surface HER2 protein expression; wherein, A: flow chart for detecting cell surface HER2 protein, B: a quantitative map;

FIG. 4 shows a graph of the results of Western immunoblotting to examine the effect of melatonin at various concentrations on HER2 positive breast cancer cells HCC1954, MDA-MB-361 and MCF7/HER2 on the signaling pathway downstream of HER 2;

FIG. 5 is a graph showing the results of Western blotting to examine the effect of melatonin and lysosomal inhibitor treatment, either single or combined, respectively, on the expression of total HER2 protein in HER2 in HER2 positive breast cancer cells HCC1954, MDA-MB-361 and MCF7/HER 2;

FIG. 6 is a graph showing the results of immunofluorescence assay of the effect of different concentrations of melatonin on co-localization of total HER2 protein and lysosomal marker LAMP1 in HER2 positive breast cancer cells HCC 1954;

FIG. 7 shows the results of melatonin and HER2 small molecule targeted inhibitor Neratinib, single or combined drug treatment, respectively, on the growth of HER2 positive breast cancer cells HCC1954, MDA-MB-361, MDA-MB453 and MCF7/HER 2;

FIG. 8 shows the results of the effect of melatonin and the HER2 small molecule targeted inhibitor, Neratinib, on HER2 positive breast cancer cells HCC1954, MDA-MB-361, MDA-MB-453 and MCF7/HER2 cell death, single or combined drug treatment, respectively, wherein A: detecting a cell death result graph by flow cytometry; b: quantifying the result;

FIG. 9 is a graph showing the results of melatonin and HER2 small molecule targeted inhibitor Lapatinib treatment on the growth of HER2 positive breast cancer cells HCC1954, MDA-MB-361, MDA-MB453 and MCF7/HER2, either singly or in combination, respectively;

FIG. 10 is a graph showing the results of melatonin and HER2 small molecule targeted inhibitor Lapatinib single or combined drug treatment on the apoptosis of HER2 positive breast cancer cells HCC1954, MDA-MB-361, MDA-MB-453 and MCF7/HER2, respectively; wherein, A: detecting an apoptosis result graph by flow cytometry; b: a quantitative result graph;

fig. 11 shows the results of melatonin and HER2 small molecule targeted inhibitors (Nratinib and Lapatinib) alone or in combination to treat the effects of HER2 positive breast cancer cells HCC1954 mouse graft tumor growth, respectively, where a: melatonin in combination with HER2 targeted inhibitor Neratinib to treat changes in the volume of transplanted tumors in mice; b: melatonin combined with HER2 targeted inhibitor Lapatinib to treat changes in the volume of transplanted tumors in mice;

figure 12 shows a graph of the results of melatonin and HER2 small molecule targeted inhibitors (Nratinib and Lapatinib) on the effects of single or combined drug treatment of HER2 positive breast cancer cells HCC1954 transplantable tumor mice on mouse body weight, where a: the effect of melatonin in combination with the HER2 targeted inhibitor Neratinib on the body weight of mice treated with transplantable tumor; b: the influence of melatonin combined with a HER2 targeted inhibitor Lapatinib on the body weight of mice treated with transplanted tumors;

fig. 13 shows microscopic observation patterns of HCC1954 parent cell and HCC1954-LapR resistant cell, in which a: HCC1954 parent cell, B: HCC1954-LapR resistant cells;

FIG. 14 is a graph showing the results of the sensitivity of HCC1954 parental cell and HCC1954-LapR resistant cell to the treatment with Lapatinib, a small molecule inhibitor of HER2, respectively;

fig. 15 shows the results of the sensitivity of HCC1954-LapR resistant cells to melatonin and the HER2 small molecule inhibitor Lapatinib, respectively, for single or combination therapy, where a: scanning a graph according to the result; b: a quantitative result chart;

FIG. 16 is a graph showing the results of HCC1954-LapR resistant cells affecting the expression of the DNA oxidative damage marker H2AX and the apoptosis marker Cleaved-PARP protein in cells treated with melatonin and the HER2 small molecule inhibitor Lapatinib, alone or in combination, respectively;

fig. 17 shows the results of the sensitivity of HCC1954 parental cells and HCC1954-LapR resistant cells, respectively, to treatment with HER2 small molecule inhibitor Neratinib, where a: scanning a graph according to the result; b: a quantitative result chart;

FIG. 18 is a graph showing the results of sensitivity of HCC1954-LapR resistant cells to melatonin and the small molecule inhibitor Neratinib, HER2, single or combination drug therapy, respectively, where A: scanning a graph according to the result; b: quantitative result chart.

Detailed Description

It should be understood that features, characteristics, components or steps described in a particular aspect, embodiment or example of the present invention may be applied to any other aspect, embodiment or example described herein unless incompatible therewith.

The foregoing disclosure generally describes the present invention, which is further illustrated by the following examples. These examples are described only to illustrate the present invention and do not limit the scope of the present invention. Although specific terms and values are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. Unless otherwise indicated, the experimental methods and techniques described herein are those well known to those skilled in the art.

Example 1 Effect of melatonin on apoptosis of HER 2-positive breast cancer cells

One, step

(1) Human HER2 positive breast cancer cells HCC1954, MDA-MB-361 and MCF7/HER2 at logarithmic growth phase were digested and counted, resuspended at 3X 10⁵Cell suspension/ml concentration 3ml of cell suspension was added to the cell culture dish, the dish was placed at 37 ℃ in 5% CO₂The culture was carried out overnight in an incubator.

(2) The following day, the fresh medium was replaced and the cells were treated with different concentrations of melatonin (0, 1, 2, 4mM) for 24, 48 hours.

(3) After the treatment, the cell supernatants of each group were collected, and the cells were digested with pancreatin without EDTA (0.25%) and collected into corresponding centrifuge tubes.

(4) Washing the cells twice with phosphate buffer Solution, diluting the 10 × Binding Solution matched with the kit to 1 × working Solution, resuspending the cells, and adjusting the cell concentration to 1 × 10⁶Ml, and 100. mu.l is taken out for use.

(5) Mu.l of PI staining solution was added to 100. mu.l of the above cell suspension, and the mixture was stained for 15 minutes at room temperature in the dark.

(6) After the incubation, 400. mu.l of 1 × Binding Solution was added, and after filtering with a 300 mesh nylon screen, detection was performed by a flow cytometer. Mapping and statistical analysis were performed using GraphPad Prism software.

Second, result in

As shown in FIG. 1, the flow cytometry results revealed that melatonin treatment induced significant increase in cell death of HER 2-positive breast cancer cells HCC1954, MDA-MB-361 and MCF7/HER2, and the tumor cell death rate increased with increasing melatonin concentration and longer duration. The experimental results show that melatonin monotherapy can induce the death of HER2 positive breast cancer cells.

Example 2 Effect of melatonin on Total HER2 protein expression in HER2 positive breast cancer cells

One, step

(1) Cells of human HER2 positive breast cancer cells HCC1954, MDA-MB-361 and MCF7/HER2 at logarithmic growth phase were digested and counted and resuspended at 3X 10⁵Cell suspension at a concentration of/ml, 3ml of the cell suspension was added to a cell culture dish, and the dish was placed at 37 ℃ with 5% CO₂The culture was carried out overnight in an incubator.

(2) The following day, the fresh medium was changed and the cells were treated for 24 hours with different concentrations of melatonin (0, 1, 2, 4 mM).

(3) After the cell treatment is finished, washing the cells twice by using a precooled phosphate buffer solution, removing a residual culture medium in a culture dish, adding a proper amount of precooled protein lysate according to the cell amount, scraping the cells by using a precooled cell scraper, transferring the cell lysate suspension into a centrifuge tube by using a micropipettor, placing the centrifuge tube on ice, paying attention to the fact that the operations are carried out on the ice as much as possible, and keeping the low-temperature operation. Inserting cell lysate containing cells into ice, placing the ice on a shaking table to lyse the cells for 15 minutes, transferring the centrifugal tube of the cell lysate containing the cells into a refrigerated centrifuge to centrifuge, wherein the centrifugation conditions are as follows: 14000 rpm, centrifugation for 15 minutes, 4 ℃. And after the centrifugation is finished, taking the supernatant in the centrifuge tube to a new centrifuge tube, and placing the centrifuge tube on ice for later use.

(4) And (3) adding the reagent and drawing a total protein standard curve by using a protein standard (BSA) provided in a rhizoxin quantification kit, taking 10 mu l of the total protein in the step (3) out of a new centrifuge tube, adding 90 mu l of pure water to dilute the protein, uniformly mixing, and placing on ice for later use. The diluted protein was added to a 96-well plate in 3 replicates per protein sample in 25 μ l per well and a blank well plate control was prepared with an equal amount of added pure water. After addition of protein and blank control, 200 μ l of mixed BCA reagent (solution a: solution B ═ 50:1) was added per well. And then placing the 96-well plate in an incubator at 37 ℃ for incubation for 30 minutes, after the incubation is finished, measuring the absorbance at 562 nm by using a multifunctional microplate reader, converting the protein concentration according to a standard curve, and multiplying by 10 times to obtain the final protein concentration. And (4) taking out a corresponding volume of protein stock solution from the residual supernatant in the step (3), diluting the protein stock solution with protein lysate to a uniform concentration, adding 4 x protein loading buffer solution with one third of the total volume, uniformly mixing, and boiling for 10 minutes at 100 ℃ by using a metal bath.

(5) And adding the protein sample with equal mass into a glue hole of the SDS-PAGE glue, and adding a protein Marker for electrophoresis. And (4) carrying out electrophoresis for about two hours at a voltage of 80V, and stopping electrophoresis after the protein marker is completely separated.

(6) And taking out the separation gel subjected to SDS-PAGE electrophoresis, soaking in a precooled electrophoresis membrane transferring liquid, fixing protein, preparing a cellulose acetate membrane with a corresponding size according to the size of the separation gel, soaking in the precooled membrane transferring liquid, and fully soaking the cellulose acetate membrane. And arranging the negative electrode (black polar plate), the sponge, the thick filter paper, the thin filter paper, the separating glue, the cellulose acetate film, the thin filter paper, the thick filter paper, the sponge and the positive electrode (transparent plate) of the rotary membrane plate in sequence according to the rotary membrane requirement. And (4) putting the film into a film transferring groove, adding a film transferring liquid, completely covering the film transferring plate, and transferring the film. And (5) rotating the membrane for 120 minutes under the condition of rotating the membrane at the voltage of 120V.

(7) After the membrane transfer, the cellulose acetate membrane was blocked with a phosphate buffer containing 5% skim milk for 1 hour at room temperature.

(8) After sealing, washing the cellulose acetate membrane with phosphate buffer solution, washing off redundant milk, cutting off the position of the cellulose acetate membrane where the target protein is located according to the indication of a protein Marker, putting the cellulose acetate membrane into an antibody incubation box, and adding diluted antibody for incubation. The incubation conditions were gentle shaking overnight on a shaker at 4 ℃.

(9) The next day, the cellulose acetate membrane was washed three times with TBST at room temperature for 10 minutes each time. After washing the primary antibody, diluted (1:5000) fluorescent secondary antibody was added and incubated for 1 hour at room temperature in the dark. After the secondary antibody incubation was complete, the cellulose acetate membrane was washed three times with TBST buffer at room temperature for 10 minutes each.

(10) Using an immunofluorescence detection System (LI-COR)

Infrared Imaging System), scan the cellulose acetate membrane, record the results and calculate the protein content.

Second, result in

As shown in FIG. 2, the results of Western blotting revealed that melatonin treatment significantly reduced the expression level of total HER2 protein in HER 2-positive breast cancer cells HCC1954, MDA-MB-361 and MCF7/HER2, and the expression level of HER2 protein was lower as the concentration of melatonin was higher. The experimental result shows that the melatonin monotherapy can effectively reduce the expression level of HER2 protein.

Example 2 Effect of melatonin on HER2 Positive Breast cancer cell surface expression of HER2 protein

One, step

(1) Human HER2 positive breast cancer cells HCC1954 in logarithmic growth phase were digested and counted, resuspended at 3X 10⁵Cell suspension/ml, 3ml of cell suspension was added to a cell culture dish, and the dish was placed at 37 ℃ with 5% CO₂The culture was carried out overnight in an incubator.

(2) The following day, the fresh medium was replaced and the cells were treated with different concentrations of melatonin (0, 2, 4mM) for 24 hours.

(3) After the drug treatment, cells were digested with 0.05% pancreatin-EDTA, after the cells were digested, the digestion was stopped with 2% FBS-containing phosphate buffer and collected into corresponding centrifuge tubes.

(4) 0.75 μ l/sample APC fluorescence isotype control, 1.25 μ l/sample APC fluorescence labeled total HER2 antibody was added to phosphate buffer containing 2% FBS for use.

(5) The cells were washed twice with 2% FBS in phosphate buffer, resuspended and stained for 15 min at room temperature in the dark.

(6) After completion of the staining, the staining was stopped with 400. mu.l of a buffer solution, filtered through a 300-mesh nylon filter, and then detected by a flow cytometer (FIG. 3A). Mapping and statistical analysis were performed using GraphPad Prism software.

Second, result in

As shown in fig. 3, it was found by flow cytometry that melatonin reduced the cell surface HER2 expression level of HER2 positive breast cancer cells HCC1954, and the cell surface HER2 protein of HCC1954 was reduced to a greater extent as the melatonin acting concentration was higher. The experimental result shows that melatonin can obviously reduce the expression level of HER2 protein on the surface of HER2 positive breast cancer cells.

Example 4 effects of melatonin on HER2 downstream signaling pathways in HER2 positive breast cancer cells

One, step

(1) Human HER2 positive breast cancer cells HCC1954 in logarithmic growth phase were digested and counted, resuspended at 3X 10⁵Cell suspension at a concentration of/ml, 3ml of the cell suspension was added to a cell culture dish, and the dish was placed at 37 ℃ with 5% CO₂The culture was carried out overnight in an incubator.

(2) The next day, the fresh medium was changed and the cells were treated with different concentrations of melatonin (0, 2, 4mM) for 12, 24, 48 hours.

The rest steps are the same as those in example 2, (3) - (10).

Second, result in

As a result, as shown in FIG. 4, it was found that Melatonin (Melatonin) decreased the protein expression of pAKT, pERK1/2, pSRC and pNF- κ B downstream of the HER2 signaling pathway in HER 2-positive breast cancer cells HCC1954 and attenuated epithelial-mesenchymal transformation (including decreased expression of N-cadherin and increased expression of E-cadherin) in Western immunoblotting. The results of this experiment indicate that melatonin may inhibit activation of the relevant oncogenic signaling pathway downstream of HER 2.

Example 5 Effect of melatonin, lysosomal inhibitor treatment alone or in combination on Total HER2 protein expression in HER2 positive breast cancer cells

Since melatonin reduced total HER2 protein expression, it was explored whether melatonin reduced total HER2 protein expression via the endocytic pathway.

One, step

(1) Human HER2 positive breast cancer cells HCC1954, MDA-MB-361 and MCF7/HER2 in logarithmic growth phase are digested and counted, resuspended in3×10⁵Cell suspension/ml concentration 3ml of cell suspension was added to the cell culture dish, the dish was placed at 37 ℃ in 5% CO₂The culture was carried out overnight in an incubator.

(2) After overnight, the fresh medium was changed and the cells were treated for 24 hours with 2mM melatonin, followed by two hours with Bafilomycin at a concentration: HCC1954 cells, MDA-MB-361 cells, 40 nM; MCF7/HER2 cells, 20 nM.

The rest steps are the same as those in example 2, (3) - (10).

Second, result in

The results are shown in fig. 5, and the western blot detection shows that a lysosome inhibitor bafilomycin (baf) can restore the reduction of HER2 protein caused by treating HER2 positive breast cancer cells HCC1954 with melatonin. The results of this experiment show that melatonin reduces the protein expression of HER2 by promoting lysosomal degradation.

Example 6 Effect of melatonin on Co-localization of Total HER2 protein and the lysosomal marker LAMP1 in HER 2-positive breast cancer cells

Since melatonin reduced total HER2 protein expression via the endocytic pathway, we determined that total HER2 was degraded via the endocytic-lysosomal pathway by examining the co-localization of total HER2 and the lysosomal marker, LAMP1, within the cell.

One, step

(1) 12-well plates were prepared and sterile coverslips were placed in each well of the plate.

(2) Human HER2 positive breast cancer cells HCC1954 in logarithmic growth phase were digested and counted, resuspended at 2X 10⁴Cell suspension at concentration of/ml, 1ml of cell suspension was added to a 12-well plate, and the 12-well plate was placed at 37 ℃ and 5% CO₂The culture was carried out overnight in an incubator.

(3) The following day, the fresh medium was changed and the cells were treated for 24 hours with different concentrations of melatonin (0, 2, 4 mM).

(4) After drug treatment, the medium was aspirated and the cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature.

(5) After cell fixation was complete, the cells were washed three times for 5 minutes each with phosphate buffer.

(6) Cells were permeabilized with phosphate buffer containing 0.1% tween-20 for 15 minutes at room temperature.

(7) After cell permeabilization, cells were washed three times for 5 minutes each with phosphate buffer.

(8) Cells were blocked with 5% BSA in phosphate buffer for 1 hour at room temperature.

(9) After cell blocking, cells were incubated overnight at 4 ℃ with mixed dilutions of total HER2 antibody and LAMP1 antibody.

(10) After the cell incubation was completed, the cells were washed three times with phosphate buffer and incubated with fluorescent secondary antibody for 1 hour at room temperature.

(11) After the secondary antibody incubation was completed, the cells were washed three times with phosphate buffer and incubated with the nuclear dye Dapi for 15 minutes.

(12) After Dapi incubation was complete, cells were washed three times with phosphate buffer and blocked on cell slides with anti-quench blocking tablets.

(13) Pictures were taken with an upright fluorescence microscope and representative pictures were taken (400 x magnification).

Second, result in

The results are shown in fig. 6, and immunofluorescence assay shows that after melatonin treatment, HER2 protein and lysosome marker LAMP1 in HCC1954 cells have a co-localization phenomenon in the cells. The experimental result shows that the melatonin is degraded through a lysosome pathway by inducing HER2 protein, so that the HER2 protein expression amount of the breast cancer cells is increased.

Example 7 Effect of melatonin and the HER2 Small molecule targeted inhibitor Neratinib, respectively, on growth of HER2 positive breast cancer cells by single or combined drug treatment

Since melatonin was able to reduce total HER2 protein expression, we examined whether melatonin could enhance the sensitivity of HER2 positive breast cancer to Neratinib treatment by reducing total HER2 protein expression.

One, step

(1) Human HER2 positive breast cancer cells HCC1954, MDA-MB-453, MDA-MB-361 and MCF7/HER2 at log phase growth were digested and counted, resuspended as a cell suspension (HCC1954 cells,MDA-MB-453 cells: 3X 10⁴Ml, MDA-MB-361 cells: 1X 10⁵MCF7/HER2 cells: 5X 10⁴/ml), 100. mu.l per well of 96-well cell culture plate was added, and the 96-well cell culture plate was placed at 37 ℃ in 5% CO₂The culture was carried out overnight in an incubator.

(2) After overnight, the cells in the first two rows of 96-well plates are stained with crystal violet, washed with a phosphate buffer solution, washed off the excess crystal violet stain as the number of the cells in zero days, the culture medium is replaced with the remaining cells, corresponding drugs are added, the cells are continuously cultured, the cells are replaced every two days, and the cells in the two rows of 96-well plates are stained with a crystal violet solution. The concentration of the medicine is as follows: HCC 1954: melatonin, 2mM, Neratinib, 50 nM. MDA-MB-453: melatonin 2mM, Neratinib, 100 nM. MDA-MB-361: melatonin, 2mM, Neratinib, 100 nM. MCF7/HER 2: melatonin, 1mM, Neratinib, 50 nM.

(3) After the cells are completely stained, re-staining all 96 holes containing the cells once by using a crystal violet solution, washing and drying, and adding 200 mu l of 50% acetic acid into each hole to dissolve the crystal violet. Quantitation was performed with a multifunctional microplate reader at 570 nm and quantitative statistical analysis was performed using Graphpad Prism software.

Second, result in

The results are shown in figure 7, and the combined drug experiment shows that compared with melatonin and HER2 small molecule targeted drug Neratinib single drug therapy, the combined drug can obviously inhibit the growth of HER2 positive breast cancer cells HCC1954, MDA-MB-453, MDA-MB-361 and MCF7/HER 2. The experimental result shows that melatonin can remarkably enhance the sensitivity of HER2 positive breast cancer to the treatment of targeted drug Neratinib.

Example 8 Effect of melatonin and the HER2 Small molecule targeted inhibitor Neratinib, respectively, on HER2 positive breast cancer cell death with single or combined drug treatment

One, step

(1) The logarithmic growth phase of human HER2 positive breast cancer cells HCC1954, MDA-MB-361, MDA-MB-453 and MCF7/HER2 were digested and counted and resuspended at 3X 10⁵Cell suspension at a concentration of/ml, 3ml of the cell suspension was added to a cell culture dish, and the dish was placed at 37 ℃ with 5% CO₂The culture was carried out overnight in an incubator.

(2) After overnight, the medium was replaced with fresh medium and the cells were treated with the desired concentration of drug for 48 hours. The concentration of the medicine is as follows: HCC 1954: melatonin, 2mM, Neratinib, 50 nM. MDA-MB-453: melatonin, 2mM, Neratinib, 100 nM. MDA-MB-361: melatonin, 2mM, Neratinib, 100 nM. MCF7/HER 2: melatonin, 2mM, Neratinib, 200 nM.

(3) After 48 hours, the cell supernatants of each group were collected and the cells were digested with pancreatin without EDTA (0.25%) and collected into corresponding centrifuge tubes.

(6) After the incubation, 400. mu.l of 1 XBinding Solution was added, filtered through a 300 mesh nylon screen and detected by a flow cytometer. Mapping and statistical analysis were performed using GraphPad Prism software.

Second, result in

As shown in the result of figure 8, the flow cytometry detection of cell death shows that compared with melatonin and HER2 small molecule targeted drug Neratinib single drug treatment respectively, the combination of the two drugs remarkably induces the death increase of HER2 positive breast cancer cells HCC1954, MDA-MB-453, MDA-MB-361 and MCF7/HER 2. The experimental result shows that Melatonin (Melatonin) can remarkably enhance the sensitivity of HER2 positive breast cancer to the treatment of a small-molecule targeted drug Neratinib.

Example 9 Effect of melatonin and the small molecule targeted inhibitor Lapatinib of HER2 on the growth of HER2 positive breast cancer cells by single or combined drug treatment, respectively

The procedure of this part of the experimental procedure was exactly the same as in example 7.

The results are shown in fig. 9, and the combined drug experiment shows that compared with melatonin and HER2 small molecule targeted drug Lapatinib single drug therapy, the combined drug can obviously inhibit the growth of HER2 positive breast cancer cells HCC1954, MDA-MB-453, MDA-MB-361 and MCF7/HER 2. The experimental result shows that Melatonin (Melatonin) can remarkably enhance the sensitivity of HER2 positive breast cancer to the treatment of a targeted drug Lapatinib.

Example 10 Effect of melatonin and the HER2 Small molecule Targeted inhibitor Lapatinib, respectively, on apoptosis in HER2 Positive Breast cancer cells with Single drug or combination drug treatment

The procedure of this part of the experimental procedure was similar to that of example 8, except that:

(1) the treatment concentrations of the drugs in the step (2) are as follows: HCC 1954: 2mM melatonin, 1. mu.M Lapatinib; MDA-MB-453: 2mM melatonin, 2. mu.M Lapatinib; MCF7/HER 2: 1mM melatonin, 1. mu.M Lapatinib; MDA-MB-361: 1mM melatonin, 2. mu.M Lapatinib.

(2) The step (5) comprises the following steps: to 100. mu.l of the above cell suspension were added 55. mu.l of PI staining solution and 5. mu.l of Annexin V-FITC dye, and the mixture was stained for 15 minutes at room temperature in the dark.

As shown in the result of figure 10, the apoptosis detection by flow cytometry shows that compared with melatonin and HER2 small molecule targeted drug Lapatinib which are respectively treated by single drug, the combination of the two drugs remarkably induces the apoptosis increase in HER2 positive breast cancer cells HCC1954, MDA-MB-453, MDA-MB-361 and MCF7/HER 2. The experimental result shows that Melatonin (Melatonin) can remarkably enhance the sensitivity of HER2 positive breast cancer to the treatment of the targeted drug Lapatinib.

Example 11 Effect of melatonin and small molecule targeted inhibitors of HER2 (Neratinib and Lapatinib) on the growth of transplanted tumors in mice with HER2 positive breast cancer cells by single or combined drug therapy, respectively

This embodiment is exemplified by the combination of Melatonin (Melatonin) and Neratinib, a small molecule targeted inhibitor of HER2, and the therapeutic regimen of Melatonin in combination with Lapatinib, a small molecule targeted inhibitor, is the same as this embodiment.

In vitro experiments, melatonin can enhance the sensitivity of HER2 positive breast cancer to treatment of Neratinib, so that the treatment effect of the melatonin combined with a HER2 targeted inhibitor (Neratinib or Lapatnib) on tumors in vivo is tested.

One, step

(1) The HCC1954 in logarithmic growth phase is thinCollecting the digested cells in a 50ml centrifuge tube, and counting the cells to ensure that the number of the cells is 1 × 10⁷The cell suspension was mixed with the thawed Matrigel gel 1:1 and mixed well, 100. mu.l of each spot was injected into each NOD/SCID mouse, two spots were injected into each mouse, and the injection site was under the breast pad of the forelimb of the mouse.

(2) After the mice are inoculated with the tumor, the tumor size of the mice is observed every day, the tumor size reaches 120 cubic millimeters (calculation method: length, width and width/2), and the mice are randomly divided into a Vehicle control group, a melatonin single treatment group, a Neratinib single treatment group and a combined treatment group. Mouse body weight was measured daily and mouse tumor size was measured every other day for 27 consecutive days of treatment. Dosage and administration mode of Neratinib: 5mg/kg/day, administration by gavage, melatonin dosage and administration mode: 50mg/kg/day, administered by intraperitoneal injection. Mouse tumor sizes were measured once a day at intervals and plotted using Graphpad Prism software for statistics.

Second, result in

The results are shown in fig. 11, the mouse transplanted tumor experiment of the HCC1954 cell line shows that the combination of the two medicines can obviously inhibit the growth of HER2 positive breast cancer cells HCC1954 xenograft mouse tumor compared with the single-medicine treatment of melatonin and HER2 small molecule targeted medicines (Neratinib and Lapatinib). The experimental result shows that the Melatonin (Melatonin) combined with HER2 small molecule targeted drugs (Neratinib and Lapatinib) can effectively inhibit the growth of mouse tumors.

Example 12 Effect of Melatonin (Melatonin) and HER2 Small molecule Targeted inhibitors (Neratinib and Lapatinib), respectively, on mouse body weight by Single or combination therapy of HER2 Positive Breast cancer cell transplantation tumor mice

Whether the drug combination is safe or not is an important index, so that the weight of mice is monitored to observe whether the drug combination has obvious toxicity or not.

First, after the start of the treatment in step (1), the body weight of mice transplanted with HER2 positive breast cancer cells HCC1954 constructed in example 11 was measured daily and recorded, and plotted using Graphpad Prism software.

Second, result in

The results are shown in fig. 12, Melatonin (Melatonin) in combination with HER2 small molecule targeted drugs (Neratinib and Lapatinib) had no significant effect on body weight of HER2 positive breast cancer cell xenograft mice. The results of this experiment show that the combination treatment regimen is not significantly cytotoxic.

Example 13 establishment of HER2 Positive Breast cancer HER2 Small molecule Targeted inhibitor drug-resistant cell line HCC1954-LapR

One, step

(1) Culturing human HER2 positive breast cancer cell strain HCC1954 in RPMI1640 medium containing 100U/mL penicillin, 100U/mL streptomycin and 10% fetal calf serum, and placing at 37 deg.C and 5% CO₂And maintaining culture in an incubator.

(2) When the cell density reached 70-90%, the supernatant was discarded and replaced with a medium containing the HER2 targeted inhibitor Lapatinib. The initial concentration of Lapatinib drug is 0.5 μ M at 37 ℃ with 5% CO₂The culture is continued under the condition, and the fresh culture medium containing Lapatinib with the same concentration is replaced every 48 hours until the cells are full.

(3) Carrying out passage on the overgrown cells in the step (2) to enable the cell density to reach about 70% after the cells adhere to the wall; continuing at 37 ℃ with 5% CO₂Culturing under the condition, replacing a fresh culture medium containing Lapatinib every 48 hours, and gradually increasing the tolerant concentration of the cell line to the Lapatinib until the cells grow well.

(4) And (3) repeating the steps (2) and (3), wherein the action concentration of the HER2 targeted inhibitor Lapatinib is gradually increased by 0.5 mu M each time until the tolerant concentration of the cell line to Lapatinib gradually reaches 5 mu M, and maintaining the cells in a culture medium containing 5.0 mu M Lapatinib for continuous expansion culture.

(5) Finally, HER2 targeted inhibitor Lapatinib resistant HCC1954-LapR cells are obtained.

(6) Screening the resulting HER2 targeted therapeutic drug resistant cells HCC1954-LapR was deposited at a second hospital affiliated with university of medical university of gangrence, address: china, Dalian.

Second, result in

As shown in FIG. 13, microscopic observation revealed that there was no significant difference in cell morphology between HCC1954 parental cell and drug-resistant HCC 1954-LapR.

Example 14 detection of the sensitivity of HCC1954 parent cell and HCC1954-LapR resistant cell to the treatment of HER2 small molecule inhibitor Lapatinib

One, step

(1) Human HER2 positive breast cancer cells HCC1954 parent cells and HCC1954-LapR resistant cells in logarithmic growth phase were digested and counted, and resuspended into cell suspension (3X 10)⁴Ml), 100. mu.l per well were added to a 96-well cell culture plate, and the 96-well cell culture plate was placed at 37 ℃ in 5% CO₂The culture was carried out overnight in an incubator. In particular, the HCC1954-LapR resistant cells were deplated 24 hours before plating, i.e. 24 hours before plating were replaced by fresh medium without Lapatinib.

(2) After overnight, the cells were replaced with medium and the corresponding drug was added, the cells were continued to be cultured, and the medium was changed every two days.

(3) After 7 days of drug treatment, the cells in each well were stained with crystal violet solution, washed and dried, and 200 μ l of 50% acetic acid was added to each well to dissolve the crystal violet. Quantification was performed using a multifunctional microplate reader at a wavelength of 570 nm and quantitative statistical analysis was performed using Graphpad Prism software.

Second, result in

The result is shown in figure 14, and the drug sensitive experiment shows that compared with HER2 positive breast cancer parent cell HCC1954, HER2 positive breast cancer drug resistant cell HCC1954-LapR has obviously reduced sensitivity to Lapatinib treatment. The experimental result shows that the drug-resistant cell HCC1954-LapR screened by the invention really has drug resistance to HER2 small molecule targeted drug Lapatinib.

Example 15 detection of the sensitivity of HCC1954-LapR resistant cells to treatment with melatonin and the HER2 small molecule inhibitor Lapatinib, alone or in combination

One, step

(1) Will grow logarithmicallyHuman HER2 positive breast cancer cells HCC1954 and HCC1954-LapR resistant cells at stage were digested, counted, resuspended as a cell suspension (1X 10)⁵Ml), 500. mu.l per well of 24-well cell culture plate was added, and the 24-well cell culture plate was placed at 37 ℃ with 5% CO₂The culture was carried out overnight in an incubator. In particular, the HCC1954-LapR resistant cells were deplated 24 hours before plating, i.e. 24 hours before plating were replaced by fresh medium without Lapatinib.

(2) After overnight, the drug was diluted to the desired concentration with complete medium and 500 μ l of drug-containing medium was added to each well, changing the medium every two days. The drug concentrations are, melatonin: 0.2, 3 mM; lapatinib: 0.2, 4. mu.M.

(3) After the Vehicle group cells were substantially full, the 24-well plate was removed from the incubator, the medium was discarded, and the plate was stained with crystal violet solution for 15 minutes at room temperature. After dyeing is finished, the 24-hole plate is cleaned and dried, and then a scanner is used for scanning the 24-hole plate and a representative picture is taken.

(4) After scanning, 500. mu.l of 50% acetic acid was added to each well to dissolve crystal violet. Quantitation was performed with a multifunctional microplate reader at 570 nm and quantitative statistical analysis was performed using Graphpad Prism software.

Second, result in

The results are shown in fig. 15, and a plate clone experiment shows that the combined administration of Melatonin (Melatonin) and HER2 small molecule targeted drug Lapatinib can effectively inhibit the growth of HER2 positive breast cancer drug-resistant cells HCC 1954-LapR. The results of this experiment indicate that the combination regimen of Melatonin (Melatonin) and Lapatinib is also effective in treating HER2 positive breast cancer resistant cells.

Example 16 Effect of HCC1954-LapR resistant cells on the expression of the DNA oxidative damage marker gamma H2AX and the apoptosis marker Cleaved-PARP protein in cells after single or combined drug treatment of melatonin and the HER2 small molecule inhibitor Lapatinib

One, step

(1) Human HER2 positive breast cancer cells HCC1954-LapR drug-resistant cells in logarithmic growth phase are digested, counted and resuspended to 3X 10⁵Cell suspension at a concentration of/ml, 3ml of cell suspension was added to a p60 cell culture dishThe cell culture dish was placed at 37 ℃ in 5% CO₂The culture was carried out overnight in an incubator. In particular, the HCC1954-LapR resistant cells were deplated 24 hours before plating, i.e. 24 hours before plating were replaced by fresh medium without Lapatinib.

(2) After overnight, the cells were treated with fresh medium containing the corresponding drug for 24 hours.

(3) The rest steps are the same as those in (3) - (10) of example 2.

Second, result in

The result is shown in fig. 16, and a protein immunoblotting experiment finds that Melatonin (Melatonin) combined with a HER2 small molecule targeted drug Lapatinib can cause that the expression of a DNA damage marker gamma H2AX and an apoptosis marker clear-PARP protein in HER2 positive breast cancer drug-resistant cells HCC1954-LapR is remarkably increased. The experimental result shows that Melatonin (Melatonin) combined with Lapatinib can effectively kill HER2 positive breast cancer drug-resistant cells.

Example 17 detection of the sensitivity of HCC1954 parental cell and HCC1954-LapR drug-resistant cell to the treatment of HER2 small molecule inhibitor Neratinib

The experimental procedure in this example is exactly the same as in example 14. In particular, the HCC1954-LapR resistant cells were deplated 24 hours before plating, i.e., 24 hours before plating were replaced with fresh medium without Lapatinib.

The result is shown in figure 17, and the drug sensitive experiment shows that compared with HER2 positive breast cancer parental cell HCC1954, HER2 positive breast cancer drug resistant cell HCC1954-LapR has obviously reduced sensitivity to Neratinib treatment. The experimental result shows that the drug-resistant cell HCC1954-LapR screened by the invention also generates drug resistance to another HER2 small molecule targeted drug Neratinib.

Example 18 testing of melatonin and the effect of Neratinib, a small molecule targeted inhibitor of HER2, alone or in combination, on the clonal development and growth of breast cancer resistant cells

The experimental procedure in this example was the same as in example 15. In particular, the HCC1954-LapR resistant cells were deplated 24 hours before plating, i.e. 24 hours before plating were replaced by fresh medium without Lapatinib.

Wherein the action concentrations of the medicines in the step (2) are respectively as follows: 0.1, 2 mM; neratinib: 0. 50, 100 nM.

The results are shown in FIG. 18, and the plate clone experiment shows that the combined administration of Melatonin (Melatonin) and HER2 small molecule targeted drug Neratinib can effectively inhibit the growth of HER2 positive breast cancer drug-resistant cells HCC 1954-LapR. The results of this experiment show that the combined regimen of Melatonin (Melatonin) and Neratinib is also effective for HER2 positive breast cancer resistant cell therapy.

Claims

1. The application of melatonin in preparing medicines for enhancing the killing effect of HER2 small molecule targeted medicines on HER2 positive breast cancer or the treatment effect on HER2 positive breast cancer; the HER2 small molecule targeted drug is lapatinib or neratinib.

2. The use of claim 1, wherein the HER2 positive breast cancer comprises HER2 positive breast cancer that is resistant or non-resistant to HER2 small molecule targeted drugs.

3. Use of melatonin and a HER2 small molecule targeted drug, the use comprising the use of any one of:

1) the application in preparing the medicine for enhancing the killing effect of the HER2 small molecule targeted medicine on HER2 positive breast cancer or the treatment effect on HER2 positive breast cancer;

2) the application in preparing the medicine for treating HER2 positive breast cancer;

the HER2 small molecule targeted drug is lapatinib or neratinib.

4. The use of claim 3, wherein HER2 positive breast cancer comprises HER2 positive breast cancer that is resistant or not resistant to HER2 small molecule targeted drugs.

5. A pharmaceutical composition for treating HER2 positive breast cancer, wherein the pharmaceutical composition comprises melatonin and a HER2 small molecule targeted drug; the HER2 small molecule targeted drug is lapatinib or neratinib.

6. The pharmaceutical composition of claim 5, wherein the HER2 positive breast cancer comprises HER2 positive breast cancer that is resistant or intolerant to HER2 small molecule targeted drugs.