Application of lutein in preparation of medicine for inhibiting breast cancer cell proliferation and medicine composition

Abstract

The invention provides application of xanthophyll in preparation of drugs for inhibiting breast cancer cell proliferation and a pharmaceutical composition for inhibiting breast cancer. The pharmaceutical composition comprises xanthophyll and cis-dichlorodiammineplatinum. The xanthophyll and cis-platinum combined use has the effect of synergistically inhibiting breast cancer cell proliferation, and the action mechanism is as follows: the xanthophyll and cis-platinum act on GSK-3beta in a targeting manner, the phosphorylation level of GSK3beta-Ser9 site is reduced, the biological activity of GSK-3beta is activated, then phosphorylation of beta-catenin is promoted, the phosphorylated beta-catenin is degraded by proteasomes, and the proliferation of breast cancer cells is inhibited. Finally, abnormal activation of a Wnt signal channel is inhibited, and the effect of inhibiting growth of breast cancer cells is achieved. The new mechanism that xanthophyll plays an anti-tumor role by regulating and controlling a GSK-3beta signal channel provides an important theoretical basis for developing breast cancer targeted therapy drugs based on natural products, provides a new drug combination scheme for treating breast cancer, is high in safety and small in side effect, and has important clinical application value and development prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of lutein in the preparation of drugs that inhibit the proliferation of breast cancer cells, and pharmaceutical compositions for inhibiting the proliferation of breast cancer cells. Background Technology

[0002] Breast cancer is one of the most common malignant tumors in women worldwide, seriously threatening women's health. Clinically, breast cancer treatment typically employs a combination of therapies, including surgery, chemotherapy, radiotherapy, and endocrine therapy, with chemotherapy being a commonly used and effective adjuvant treatment. Cisplatin is a commonly used chemotherapy drug for breast cancer; however, the emergence of drug resistance or dose tolerance significantly impacts its efficacy. Therefore, identifying new therapeutic targets and novel combination therapy regimens, as well as developing highly effective and low-toxicity anti-breast cancer drugs, is of great significance for breast cancer treatment.

[0003] The Wnt signaling pathway (a multi-downstream signal transduction pathway) is a highly conserved signal transduction mechanism throughout biological evolution. Numerous studies have shown that the classical Wnt (Wnt / β-catenin) pathway plays a crucial role in the entire process of breast cancer development by regulating the expression of related signaling proteins and genes, and is currently considered an important target for breast cancer treatment. GSK-3β is a key enzyme upstream of the Wnt pathway; it exerts its tumor-suppressive effect by promoting β-catenin phosphorylation, which leads to its degradation by the proteasome, thereby inhibiting the Wnt signaling pathway.

[0004] This study found that lutein can inhibit breast cancer cell proliferation and increase cell sensitivity to the therapeutic drug cisplatin. The combination regimen significantly enhanced the inhibitory activity against breast cancer cells compared to lutein or cisplatin alone. Further research revealed that lutein can inhibit phosphorylation of the key Wnt pathway protein GSK3βSer9, promote β-Catenin degradation, block abnormal activation of the Wnt pathway, and induce apoptosis, thereby promoting tumor cell proliferation. Currently, there are no studies or reports on the application of lutein in the treatment of breast cancer in existing technologies. Therefore, this invention proposes the application of lutein in inhibiting breast cancer cell proliferation and provides a pharmaceutical composition for inhibiting breast cancer cell proliferation, which is of great significance for the treatment of breast cancer patients and the development of new treatment strategies. Summary of the Invention

[0005] This invention addresses the problems of single drug target and significant chemotherapy resistance in existing breast cancer treatments by proposing the application of lutein in the preparation of drugs that inhibit breast cancer cell proliferation, and provides a pharmaceutical composition for inhibiting breast cancer cell proliferation, thus offering a new option for breast cancer treatment.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: (i) This invention provides the application of lutein in the preparation of drugs that inhibit the proliferation of breast cancer cells. Lutein can act on the GSK-3β target site, reduce the phosphorylation level of the GSK3β-Ser9 site, activate its activity, promote β-catenin phosphorylation so that it is degraded by the proteasome, thereby inhibiting the Wnt signaling pathway and exerting the activity of inhibiting the growth of breast cancer cells.

[0007] (ii) The present invention also provides a pharmaceutical composition for inhibiting the proliferation of breast cancer cells, the pharmaceutical composition comprising lutein and cis-dichlorodiamineplatin.

[0008] As a further preferred embodiment of the present invention, the mass ratio of lutein to cis-dichlorodiammine platinum in the pharmaceutical composition is 20-50:1. As a further preferred embodiment of the present invention, the mass ratio of lutein to cis-dichlorodiamineplatinum in the pharmaceutical composition is 25:1.

[0009] As a further preferred embodiment of the present invention, in the pharmaceutical composition, lutein can, on the one hand, inhibit the phosphorylation of GSK-3β and activate its activity, thereby downregulating the expression of β-catenin and inhibiting the growth of breast cancer cells; on the other hand, lutein can effectively improve cisplatin resistance and thus inhibit the growth of breast cancer cells.

[0010] As a further preferred embodiment of the present invention, the pharmaceutical composition further includes pharmaceutical excipients; the pharmaceutical excipients include one or more of fillers, binders, disintegrants, lubricants, flow aids, coating materials, solvents, preservatives, and sustained-release materials.

[0011] As a further preferred embodiment of the present invention, the dosage form of the pharmaceutical composition is one or more of tablets, granules, capsules, solutions, emulsions, ointments, and injections.

[0012] As a further preferred embodiment of the present invention, the breast cancer is Luminal breast cancer or triple-negative breast cancer.

[0013] (iii) The present invention also provides the application of the above-described pharmaceutical composition in inhibiting the proliferation of breast cancer cells.

[0014] The beneficial effects of this invention are: (1) This invention proposes the application of lutein in inhibiting the proliferation of breast cancer cells. It is the first time that lutein can act on the GSK-3β target and inhibit its activity, thereby inhibiting the growth of breast cancer cells and providing a new idea for the development of novel anti-breast cancer drugs. (2) This invention provides a pharmaceutical composition comprising lutein and cis-dichlorodiamineplatin. Lutein, on the one hand, can inhibit the phosphorylation of GSK-3β at the Ser9 site, thereby activating its activity and promoting the phosphorylation and degradation of downstream substrates such as β-catenin, ultimately inhibiting the proliferation of breast cancer cells. On the other hand, lutein can work synergistically with cisplatin to produce a synergistic anti-tumor effect, enhancing the inhibitory effect on the proliferation of breast cancer cells, thus providing a new combination therapy for the treatment of breast cancer. (3) The lutein provided by this invention is a natural compound with advantages such as high safety and few side effects, which is of great significance for the development of highly effective and low-toxicity anti-breast cancer drugs. Attached Figure Description

[0015] Figure 1 This is a graph showing the inhibitory activity of lutein and cisplatin, alone or in combination, on the proliferation of breast cancer cells MDA-MB-468 and MCF-7. Figure 2 A diagram showing apoptosis in MCF-7 and MDA-MB-468 breast cancer cells after lutein treatment; Figure 3 The relative expression levels of GSK-3β, Phospho-GSK-3β, and β-catenin proteins after lutein treatment of MCF-7 breast cancer cells; Figure 4 The values ​​represent the relative expression levels of GSK-3β, Phospho-GSK-3β, and β-catenin proteins in MDA-MB-468 breast cancer cells after lutein treatment. Detailed Implementation To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the embodiments and accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0016] The present invention provides a pharmaceutical composition for inhibiting the proliferation of breast cancer cells, wherein the breast cancer is Luminal breast cancer or triple-negative breast cancer, and the pharmaceutical composition comprises lutein and cis-dichlorodiamine platinum, wherein the mass ratio of lutein to cis-dichlorodiamine platinum is 20 to 50:1, more preferably 25:1.

[0017] In the above-mentioned drug composition, lutein can inhibit the growth of breast cancer cells by inhibiting phosphorylation at the GSK-3βSer9 site, activating enzyme activity, and ultimately inhibiting the growth of breast cancer cells. On the other hand, the combined use of lutein and cisplatin can effectively enhance the inhibitory activity against breast cancer cell proliferation.

[0018] Furthermore, the pharmaceutical compositions of the present invention also include one or more of the following: fillers, binders, disintegrants, lubricants, flow aids, coating materials, solvents, preservatives, and sustained-release materials. The dosage form of the pharmaceutical compositions may be one or more of the following: tablets, granules, capsules, solutions, emulsions, ointments, and injections.

[0019] The following is an efficacy verification of lutein and the drug combination.

[0020] Example 1: Effects of lutein and cisplatin on breast cancer cell proliferation The effects of lutein, cisplatin, and the combination of lutein and cisplatin on the proliferation of breast cancer cells MDA-MB-468 and MCF-7 were detected using the CCK-8 assay.

[0021] Experimental methods: Logarithmic growth phase breast cancer cells MDA-MB-468 and MCF-7 were seeded in 96-well plates with 100 μL of culture medium per well; the drug was diluted with culture medium to set multiple concentrations; 100 μL of drug-containing culture medium was added to each well, and the cells were cultured for 72 / 120 hours; 10 μL of CCK-8 solution was added to each well, gently mixed, and incubated at 37°C in the dark for 1-4 hours; the OD value at 450 nm was measured using a microplate reader; cell viability was calculated based on the OD value.

[0022] Test results are as follows Figure 1 As shown in the figure, the combination of different concentrations of lutein and cisplatin exhibited superior inhibitory activity compared to cisplatin alone. The 25 μM lutein-cisplatin combination showed the most significant inhibitory effect, and the difference between its inhibition curve and that of the cisplatin-only group increased with increasing cisplatin concentration. These results indicate that lutein and cisplatin have a synergistic inhibitory effect on the proliferation of luminal MCF-7 and triple-negative MDA-MB-468 breast cancer cells, and this effect is concentration-dependent on lutein.

[0023] Example 2: Lutein promotes apoptosis in breast cancer cells The effect of lutein on apoptosis in MCF-7 and MDA-MB-468 cells was detected using the Annexin V-FITC / PI apoptosis kit. The experimental methods are as follows: 1×10 6 / cell volume, when cell confluence reaches 70-80%, treat cells with lutein for 6 hours. Wash cells once in PBS, then once in 1X Binding Buffer. Transfer cells at 1-5 x 10⁻⁵ cells / well. 6Resuspend the cells in 1X binding buffer at 1 mL / mL, add 5 μL Annexin V to 100 μL of cell suspension, incubate at room temperature for 10-15 minutes, wash the cells with 1X Binding Buffer, resuspend the cells in 200 μL of 1X Binding Buffer, add 5 μL of 7-AAD active staining solution, and analyze by flow cytometry.

[0024] Test results are as follows Figure 2 As shown in the figure. Annexin V-FITC / PI double-staining flow cytometry was used to detect the apoptosis-inducing effect of lutein on MCF-7 (ER-positive) and MDA-MB-468 (triple-negative) breast cancer cells. The results showed that lutein treatment significantly increased the proportions of early apoptosis and late apoptosis / necrosis in both MCF-7 and MDA-MB-468 cells, suggesting that lutein can effectively induce apoptosis in two different molecular subtypes of breast cancer cells.

[0025] Example 3: Lutein inhibits phosphorylation at the GSK-3βSer9 site and promotes the degradation of β-catenin. The effects of lutein on the expression of Wnt pathway proteins such as GSK-3β and β-catenin in MCF-7 and MDA-MB-468 cells were detected by Western blotting. The experimental methods are as follows: 1. Cell Culture and Drug Treatment MCF-7 and MDA-MB-468 cells were revived and cultured separately in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics. Cells were passaged routinely in a 37°C, 5% CO2 incubator, and cells in the logarithmic growth phase were selected for experiments. The cell density for both types of cells was adjusted to 5 × 10⁻⁶ cells / year. 5 Cells were seeded per well in 6-well cell culture plates, with 2 mL of complete culture medium added to each well. Cells were cultured for 24 h to allow adherence. A control group and a lutein treatment group were set up: the control group received an equal volume of culture medium (containing DMSO as a drug dissolution solvent, final concentration ≤0.1% to avoid solvent toxicity), while the lutein treatment group received culture medium containing different concentrations of lutein. After culturing for another 48 h, the culture medium in the wells was discarded, and the cells were gently washed 2-3 times with pre-cooled PBS buffer for 5 min each time to remove residual culture medium and serum.

[0026] 2. Extraction of total cell protein Every 10 6Add approximately 120 µL of RIPA lysis buffer (add protease inhibitor and phosphatase inhibitor a few minutes before use) to the culture plate / flask and lyse for 3-5 minutes. During this time, repeatedly shake the culture plate / flask to ensure thorough contact between the reagents and cells. Use a cell scraper to scrape off the cells and transfer the lysis buffer to a pre-chilled 1.5 mL centrifuge tube. Lyse on ice for 15 minutes, repeatedly pipetting to ensure complete cell lysis. Centrifuge at 12500 rpm, 4 °C for 15 minutes. Collect the supernatant (total protein extract) into a new centrifuge tube and discard the precipitate (cell debris).

[0027] 3. SDS-PAGE gel electrophoresis The protein concentration was determined according to the BCA protein concentration assay kit instructions. The protein solution was added to 5×SDS loading buffer at a 4:1 ratio, denatured in a boiling water bath for 7 min, and stored at -80℃ for later use. A 10% separating gel and a 5% stacking gel were prepared (GSK-3β protein has a molecular weight of approximately 47 kDa; a 10% separating gel can achieve good separation). After the gel solidified, it was placed in an electrophoresis tank, and Tris-glycine electrophoresis buffer was added. Protein marker (5 μL) and the denatured sample (20-30 μL per lane) were added sequentially. Electrophoresis was first performed at a constant voltage of 80V until the sample entered the separating gel (approximately 30 min). The voltage was then adjusted to 120V, and electrophoresis was continued until the bromophenol blue indicator reached approximately 1 cm from the bottom of the gel. Electrophoresis was then terminated, and the sample was transferred to a membrane.

[0028] 4. Transfer printing (wet transfer printing) Prepare two thick filter papers and a PVDF membrane of appropriate size. The PVDF membrane should be activated with methanol for 2 minutes before use. Assemble the transfer sandwich in the following order: sponge pad → filter paper → gel → PVDF membrane → filter paper → sponge pad (be careful to remove air bubbles and avoid misalignment of the gel and membrane). Place it in the transfer tank, ensuring that the PVDF membrane faces the positive electrode and the gel faces the negative electrode. Transfer at a constant current of 200mA for 90 minutes at 4℃ (or adjust according to the protein molecular weight; for 47kDa protein, the transfer time can be set to 80-100 minutes). After the transfer is complete, stain the PVDF membrane with Ponceau S for 5 minutes and observe whether the protein marker bands are clearly transferred. After verifying the transfer effect, wash off the stain with TBST buffer.

[0029] 5. Blocking and antibody incubation After transfer, place the membrane in an incubator containing TBST, rinse quickly once, then add 5% skim milk and place on a decolorizing shaker for blocking at room temperature for 30 minutes. Following the antibody instructions, dilute the primary antibody with 5% skim milk. After preparation, add the prepared primary antibody to the membrane and incubate overnight at 4°C. The next day, warm to room temperature on a shaker for 1 hour. The following day, discard the primary antibody solution and wash the membrane three times with 1X TBST buffer for 5 minutes each time to remove unbound primary antibody.

[0030] Dilute the corresponding secondary antibody with 5% skim milk at a ratio of 1:5000. Place the membrane in the secondary antibody solution and incubate on a shaker at room temperature for 30 minutes. Wash the membrane three times with 1XTBST for 5 minutes each time. Mix equal volumes of ECLA and ECLB solutions in a centrifuge tube. Place a self-sealing bag on a gel imaging plate, place the PVDF membrane protein side up on the self-sealing bag, add the mixed ECL solution, and allow it to react fully. After 1-2 minutes, place it in the gel imaging system for automatic exposure to acquire protein band images.

[0031] Test results are as follows Figure 3 and Figure 4 As shown, lutein treatment decreased p-GSK3β (Ser9) levels in both MCF-7 and MDA-MB-468 cells, while total GSK3β protein remained largely unchanged, suggesting that lutein activates GSK3β's biological activity by inhibiting phosphorylation at the Ser9 site. Simultaneously, the expression of β-Catenin in the Wnt pathway was significantly downregulated, an effect consistent with that of the Wnt pathway inhibitor XAV939. This indicates that lutein promotes β-Catenin degradation and blocks abnormal activation of the Wnt pathway by targeting and regulating GSK3β phosphorylation.

[0032] This invention is the first to discover that lutein can induce apoptosis in two subtypes of breast cancer cells: luminal MCF-7 and triple-negative MDA-MB-468, and inhibit tumor cell proliferation. When used in combination with the traditional anti-tumor drug cisplatin, it exhibits synergistic anti-tumor activity. Mechanistic studies revealed that lutein inhibits breast cancer cell growth by regulating the phosphorylation level of the key protein GSK-3β, thereby downregulating the activity of the Wnt signaling pathway. This invention clarifies the potential application value of lutein as a Wnt pathway regulator, providing new insights for finding novel anti-breast cancer drugs and developing new anti-tumor combination therapy strategies, and has significant clinical application value. Furthermore, the combined use of lutein may reduce the effective concentration of cisplatin, providing experimental evidence for reducing the clinical dosage of cisplatin and mitigating its toxic side effects.

[0033] The above are merely preferred embodiments of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principle of the present invention should be considered within the scope of protection of the present invention.

Claims

1. Application of lutein in the preparation of drugs that inhibit the proliferation of breast cancer cells.

2. Application of lutein in the preparation of drugs that inhibit breast cancer cell proliferation in combination with cisplatin.

3. A pharmaceutical composition for inhibiting the proliferation of breast cancer cells, characterized in that, The pharmaceutical composition comprises lutein and cis-dichlorodiamineplatinum.

4. The pharmaceutical composition according to claim 2, characterized in that, In the pharmaceutical composition, the mass ratio of lutein to cis-dichlorodiamine platinum is 20-50:

1.

5. The pharmaceutical composition according to claim 2, characterized in that, In the pharmaceutical composition, the mass ratio of lutein to cis-dichlorodiamine platinum is 25:

1.

6. The pharmaceutical composition according to claim 2, characterized in that, The pharmaceutical composition inhibits the growth of breast cancer cells by inhibiting the phosphorylation of GSK-3β and activating its activity.

7. The pharmaceutical composition according to claim 2, characterized in that, The pharmaceutical composition also includes pharmaceutical excipients; The pharmaceutical excipients include one or more of the following: fillers, binders, disintegrants, lubricants, flow aids, coating materials, solvents, preservatives, and sustained-release materials.

8. The pharmaceutical composition according to claim 2, characterized in that, The dosage form of the pharmaceutical composition is one or more of the following: tablets, granules, capsules, solutions, emulsions, ointments, and injections.

9. The pharmaceutical composition according to claim 2, characterized in that, The breast cancer mentioned is Luminal breast cancer or triple-negative breast cancer.

10. The use of the pharmaceutical composition according to any one of claims 2 to 8 in inhibiting the proliferation of breast cancer cells.

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