An antitumor agent and its application

Abstract

This application belongs to the field of biomedical technology. This application discloses an antitumor agent and its application. The antitumor agent of this application comprises: (a) rosiglitazone; (b) one or more inhibitors for reducing the expression of fructose-1,6-bisphosphate aldolase C. The antitumor agent of this application can more effectively inhibit the proliferation of tumor cells and can be used as a potential antitumor agent.

Description

Technical Field

[0001] This application belongs to the field of biomedical technology, and specifically relates to an antitumor agent and its application. Background Technology

[0002] Tumor metabolic reprogramming is a key characteristic of tumor cells, typically manifested as alterations in metabolic pathways such as enhanced glycolysis and abnormal oxidative phosphorylation. In recent years, therapeutic strategies targeting tumor metabolic reprogramming have received widespread attention, with glycolysis inhibitors (such as 2-deoxy-D-glucose, 2-DG) showing some anti-tumor potential in preclinical studies. However, these strategies generally face challenges such as poor selectivity, significant toxic side effects, and drug resistance. For example, while 2-DG can inhibit glycolysis, it also interferes with the metabolism of normal cells in the process, leading to adverse reactions such as neurotoxicity and hypoglycemia, thus limiting its clinical application.

[0003] Rosiglitazone (Rosi) is a second-generation thiazolinone (TZD) drug, a full agonist of peroxisome proliferator-activated receptor gamma (PPARγ), primarily used clinically to treat type 2 diabetes. Long-term use of Rosi may lead to adverse reactions such as weight gain, edema, and heart failure. Recent studies have shown that Rosi also has potential anti-tumor effects; however, its anti-tumor activity is influenced by various factors, and its clinical application is severely limited by side effects and potential safety concerns, hindering its further use in cancer treatment. Fructose-1,6-bisphosphate aldolase C (ALDOC) is an important enzyme in the glycolysis pathway, catalyzing the breakdown of fructose-1,6-bisphosphate into dihydroxyacetone phosphate and glyceraldehyde-3-phosphate. Its high expression in various tumor types has been widely confirmed, and this high expression is closely related to tumor metabolic reprogramming, proliferation, migration, invasion, and the maintenance of tumor stem cell characteristics.

[0004] Current treatments for tumor metabolism suffer from limitations such as insufficient selectivity, significant side effects, and limited efficacy. A key technical challenge is how to target key molecules in tumor metabolism to reduce tumor survival activity and synergistically enhance the anti-tumor activity of PPARγ agonists. Summary of the Invention

[0005] Objective of the Invention: This application provides an antitumor preparation and its application. The antitumor preparation of this application enhances the antitumor effect of PPARγ agonists through combined metabolic regulation, overcoming the problems of poor selectivity, large toxic side effects, and limited efficacy of existing antitumor drugs, thereby improving the antitumor effect and reducing adverse drug reactions.

[0006] Technical solution: This application provides an antitumor preparation, comprising:

[0007] (a) Rosiglitazone;

[0008] (b) One or more inhibitors for reducing the expression of fructose-1,6-bisphosphate aldolase C (ALDOC).

[0009] The antitumor preparation of this application includes an inhibitor for reducing the expression of fructose-1,6-bisphosphate aldolase C (ALDOC), which can significantly inhibit the proliferation of cancer cells. Data from the examples in this application show that the combined use of rosiglitazone and the inhibitor for reducing fructose-1,6-bisphosphate aldolase C (ALDOC) expression can significantly enhance its antitumor effect while ensuring safety.

[0010] In some embodiments, the inhibitors for reducing fructose-1,6-bisphosphate aldolase C expression include one or more inhibitors for reducing the expression levels of fructose-1,6-bisphosphate aldolase C gene, fructose-1,6-bisphosphate aldolase C mRNA, and fructose-1,6-bisphosphate aldolase C protein in tumor cells.

[0011] In some embodiments, the inhibitor for reducing fructose-1,6-bisphosphate aldolase C expression comprises one or more of siRNA, shRNA, and sgRNA targeting the fructose-1,6-bisphosphate aldolase C gene sequence or the fructose-1,6-bisphosphate aldolase C mRNA sequence, or is nanoparticles, viral vectors, PEG-modified proteins, protein microspheres, liposomes, or extracellular vesicles carrying siRNA, shRNA, or sgRNA targeting the fructose-1,6-bisphosphate aldolase C mRNA sequence.

[0012] In some embodiments, the inhibitor for reducing fructose-1,6-bisphosphate aldolase C expression comprises shRNA targeting the fructose-1,6-bisphosphate aldolase C mRNA sequence, the shRNA sequence including the sequence shown in SEQ ID NO:7 or SEQ ID NO:8.

[0013] In some embodiments, the CDS sequence of the fructose-1,6-bisphosphate aldolase C is shown in SEQ ID NO:13.

[0014] In some embodiments, the shRNA is constructed in a plasmid vector.

[0015] In some embodiments, the backbone structure of the plasmid vector is a pLKO.1 vector.

[0016] In some embodiments, the dosage of rosiglitazone is 0.5 mg / kg / d to 40 mg / kg / d, preferably 10 mg / kg / d to 20 mg / kg / d.

[0017] In some embodiments, the antitumor agents described in this application further include palmitic acid.

[0018] In some embodiments, the antitumor preparation further comprises one or more of pharmaceutically or immunologically acceptable excipients, carriers, and diluents.

[0019] In some embodiments, the antitumor agents described in this application comprise palmitic acid and rosiglitazone.

[0020] In some embodiments, the antitumor agents described in this application comprise: palmitic acid, and one or more inhibitors for reducing the expression of fructose-1,6-bisphosphate aldolase C (ALDOC).

[0021] Other embodiments of this application provide the use of the described antitumor agents in the preparation of antitumor drugs.

[0022] In some embodiments, the tumor is breast cancer.

[0023] Beneficial effects: (1) Enhanced anti-tumor effect: The application uses rosiglitazone and an ALDOC-targeting inhibitor to inhibit ALDOC and activate PPARγ, which can significantly enhance the anti-tumor effect. In in vitro experiments, the cell proliferation inhibition rate of MCF7 cells increased to 70.4% and the Bliss synergistic score reached 28.3%. In BT549 cells, the cell proliferation inhibition rate increased to 65.6% and the Bliss synergistic score reached 23.8%, indicating that there is a significant synergistic anti-tumor effect between the two, which can effectively enhance the anti-tumor effect of rosiglitazone. The application also verified the significant tumor inhibition effect in in vivo animal models. In the mouse EO771 subcutaneous tumor model, the tumor volume of the combined treatment group was only 297±27. mm³, which is about 75.6% lower than the control group, and the growth inhibition rate of tumor volume is significantly higher than that of the rosiglitazone group and the ALDOC knockdown group; (2) Reduce drug side effects: This application enhances the activity of rosiglitazone by inhibiting the expression of ALDOC, which can reduce the required dose of rosiglitazone, thereby helping to reduce adverse reactions such as weight gain caused by higher doses of rosiglitazone; (3) Precise targeting and reduction of unnecessary side effects: The scheme of this application can precisely target the expression of ALDOC in tumor cells and reduce the impact on normal cells. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 This is a diagram illustrating the tumor-suppressing mechanism of the proposed solution;

[0026] Figure 2 The results of Aldoc knockdown RT-qPCR in mouse EO771 cells in this application are shown.

[0027] Figure 3 The following are Western Blot results of Aldoc knockdown of mouse EO771 cells in the embodiments of this application;

[0028] Figure 4 The tumor shape of subcutaneous EO771 cells after Aldoc knockdown in the embodiments of this application;

[0029] Figure 5 In this application embodiment, the weight of subcutaneous tumor cells formed by Aldoc knockout of mouse EO771 cells was reduced.

[0030] Figure 6 The results of the changes in the volume of subcutaneous tumors formed by Aldoc knockdown in mouse EO771 cells in the embodiments of this application;

[0031] Figure 7 The results of RT-qPCR for knockdown of human breast cancer cells MCF7 and BT549 ALDOC in the embodiments of this application;

[0032] Figure 8 The results of Western Blot analysis of human breast cancer cells MCF7 and BT549 ALDOC knockdown in the embodiments of this application;

[0033] Figure 9 The results of CCK8 knockdown in human breast cancer cells MCF7 and BT549 ALDOC in the embodiments of this application;

[0034] Figure 10 The CCK8 results for human breast cancer cells MCF7 and BT549 ALDOC knockdown combined with different concentrations of rosiglitazone in the embodiments of this application;

[0035] Figure 11 The results of the combined treatment of human breast cancer cells MCF7 and BT549 with ALDOC KD1 and rosiglitazone (10 μmol / L) in the embodiments of this application are as follows;

[0036] Figure 12 These are gross images of subcutaneous tumor formation caused by EO771 Aldoc knockdown combined with rosiglitazone in the embodiments of this application. In these images, shNC+DMSO represents the experimental results of the relative control group, shNC+Rosi represents the experimental results of the control plasmid and rosiglitazone, shNC+PA represents the experimental results of the control plasmid and palmitic acid combination, shNC+Rosi+PA represents the experimental results of the control plasmid and rosiglitazone and palmitic acid combination, Aldoc KD1+DMSO represents the experimental results of the Aldoc knockdown group, Aldoc KD1+Rosi represents the Aldoc knockdown and rosiglitazone combination group, Aldoc KD1+PA represents the experimental results of the Aldoc knockdown and palmitic acid combination, and Aldoc KD1+Rosi+PA represents the Aldoc knockdown and rosiglitazone and palmitic acid combination group.

[0037] Figure 13 The results of the mouse EO771 Aldoc knockout subcutaneous tumor weight reduction experiment in the embodiments of this application;

[0038] Figure 14 The results of the mouse EO771 Aldoc knockout subcutaneous tumor volume reduction experiment in the embodiments of this application;

[0039] Figure 15 The CO-IP experiment in the embodiments of this application demonstrates the experimental results of HSP90 combined with ALDOC;

[0040] Figure 16 The CO-IP experiment in the embodiments of this application demonstrates the experimental results of HSP90 binding to PPARγ;

[0041] Figure 17 The competitive CO-IP experiment in the embodiments of this application demonstrates the experimental results of ALDOC and PPARγ competitively binding to HSP90;

[0042] Figure 18 The gradient transfection of ALDOC in the embodiments of this application demonstrates the experimental results of ALDOC competitively binding to HSP90 with PPARγ. Detailed Implementation

[0043] The technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application. In addition, in the description of this application, the term "comprising" means "including but not limited to". The terms first, second, third, etc. are used only as illustrative purposes and do not impose numerical requirements or establish an order. Various embodiments of this application may exist in the form of a range; it should be understood that the description in the form of a range is only for convenience and conciseness and should not be construed as a hard limitation on the scope of this application; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single values ​​within that range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which applies regardless of the range. Additionally, whenever a range of numbers is specified in this document, it means including any referenced numbers (fractions or integers) within the range referred to.

[0044] While rosiglitazone has shown some antitumor activity in certain cancers, its widespread clinical application is limited by severe side effects and limited efficacy. Currently, ALDOC, a key enzyme in tumor metabolic reprogramming, is highly expressed in various tumors, promoting tumor proliferation, migration, and drug resistance. Therefore, combination therapy combining ALDOC inhibition with rosiglitazone may offer a new approach to overcome the limitations of single-target therapy and improve treatment outcomes. Figure 1 As shown, this application proposes to enhance the anti-tumor effect by combining an ALDOC inhibitor and rosiglitazone, thereby solving the problems of limited efficacy and excessive side effects of rosiglitazone in the prior art, and providing a novel combination therapy strategy aimed at more effectively inhibiting the proliferation of tumor cells and overcoming the limitations of existing treatment methods.

[0045] Example 1: Knockdown of Aldoc gene expression in mouse cancer cells

[0046] Mouse Aldoc shRNA plasmids were used to transfect mouse breast cancer cells EO771 to establish a stable cell model with knocked-down Aldoc expression. Two different Aldoc shRNA plasmid sequences (Aldoc-shRNA1 and Aldoc-shRNA2) were used for transfection experiments. The specific steps are as follows:

[0047] (1) EO771 cells were loaded with 5 × 10 4Prepare a suspension at a concentration of 100 μL / mL, inoculate 100 μL into each well of a 96-well plate, and incubate for 48 h until the confluence reaches 60%–70%.

[0048] (2) Take out the mouse Aldoc shRNA plasmid (Aldoc shRNA plasmid vector is pLKO.1 backbone, including Aldoc-shRNA1 and Aldoc-shRNA2, purchased from Hunan Fenghui Biotechnology Co., Ltd.) stored in a low temperature environment and thaw it slowly on ice;

[0049] (3) Add 100 ng of mouse Aldoc shRNA plasmid, 5 μL of serum-free culture medium and 0.16 μL of Lipo8000 transfection reagent to the well to make a mixture. After mixing, let stand for 15 min and then add to the cell well. Add 100 μL of complete culture medium and replace with complete culture medium after 6 h.

[0050] (4) Continue culturing for 2-3 days, during which time the nutrient medium can be supplemented or replaced to maintain cell viability;

[0051] (5) Confirmation of transfection effect: 48 hours after transfection, samples were collected and the knockdown efficiency was observed by quantitative PCR (RT-qPCR), and the protein expression changes were verified by Western Blot. The mRNA and protein expression were significantly inhibited, indicating that the cell line was successfully transfected.

[0052] The two nucleotide sequences of the Aldoc shRNA are shown below (both have the pLKO.1 plasmid vector as their backbone):

[0053] Aldoc-shRNA1: 5′-GCCAGTCTTGATCAGGACTTT-3′ (SEQ ID NO: 1)

[0054] Aldoc-shRNA2: 5′-GGAATGAGTGGTCTTCAGGAA-3′ (SEQ ID NO: 2)

[0055] Each shRNA was synthesized and cloned into the pLKO.1 vector, which was then used to transfect mouse breast cancer cells EO771 to achieve knockdown of the Aldoc gene.

[0056] To verify the knockdown effect, the experimental cells were divided into three groups:

[0057] (1) Relative control group (shNC): Mouse breast cancer cells EO771 transfected only with the control plasmid shNC and not transfected with any shRNA plasmid served as the negative control group;

[0058] (2) Aldoc-shRNA1 group (Aldoc KD1): EO771 cells transfected with pLKO.1 plasmid containing Aldoc-shRNA1 (SEQ ID NO:1);

[0059] (3) Aldoc-shRNA2 group (Aldoc KD2): EO771 cells transfected with pLKO.1 plasmid containing Aldoc-shRNA2 (SEQ ID NO:2).

[0060] After transfection, the three groups of cells were treated under the same culture conditions for 48 hours. Samples were collected, and the mRNA expression level of the Aldoc gene was detected using RT-qPCR. Changes in Aldoc protein expression were detected by Western blotting to verify the knockdown effect of Aldoc in breast cancer cells EO771. Results are as follows: Figure 2 As shown, compared with the relative control group, both the Aldoc-shRNA1 group and the Aldoc-shRNA2 group significantly reduced the expression level of Aldoc, verifying the effectiveness of shRNA plasmid construction.

[0061] The specific steps for the RT-qPCR method are as follows:

[0062] (1) Using RNA extraction reagent (Trizol), cells from the control group, Aldoc-shRNA1 group and Aldoc-shRNA2 group were collected, and total RNA was extracted and reverse transcription was performed;

[0063] (2) Real-time quantitative PCR, primer sequences:

[0064] Aldoc-Forward: 5′-TGGCTGAGTATAGGTAAGGT-3′ (SEQ ID NO: 3)

[0065] Aldoc-Reverse: 5′-AGGCTGAGTGAGTGGATT-3′ (SEQ ID NO:4)

[0066] Actb-Forward: 5′-AGAGGGAAATCGTGCGTGAC-3′ (SEQ ID NO: 5)

[0067] Actb-Reverse: 5′-CAATAGTGATGACCTGGCCGT-3′ (SEQ ID NO: 6)

[0068] (3) Prepare the reagents according to Table 1, operate on ice, and mix well;

[0069] Table 1. Reagent volumes used in RT-qPCR

[0070]

[0071] (4) Add the mixed liquid to the real-time PCR plate. Seal the real-time PCR plate with sealing film, protect it from light, and centrifuge briefly.

[0072] The Ct value of each well was detected using the StepOne software, and the relative expression level (2^-ΔΔCt) was calculated using Actb as an intrinsic parameter.

[0073] The results are as follows Figure 2 The results showed that Aldoc gene expression was significantly reduced in the transfected group, indicating that the shRNA plasmid had a good interference effect on the target gene. The expression level of Aldoc protein was detected by Western blotting, and the results are as follows: Figure 3 As shown, from Figure 3 The results show that the shRNA plasmid has a good interference effect on the expression level of Aldoc protein.

[0074] Based on the combined results of RT-qPCR and Western Blot, Aldoc-shRNA1 showed higher knockdown efficiency, so it was selected for subsequent experiments.

[0075] To further investigate the effects of Aldoc knockdown on tumorigenesis and development, a subcutaneous tumor model in C57BL / 6 mice was constructed using a stable EO771 cell line transfected with the Aldoc-shRNA1 plasmid. Six- to eight-week-old female C57BL / 6 mice were used in the experiment, and the cells were administered at a rate of 5 × 10⁴ cells per mouse. 5 A dose of [number] mice was suspended in 100 μL of PBS solution and injected subcutaneously into the right axilla. The control group was injected with EO771 cells transfected with the control plasmid. Five mice were included in each of the experimental and control groups.

[0076] Every two days after injection, the long and short diameters of the tumor were measured using calipers, and the tumor volume (unit: mm³) was calculated using the formula V = (long × short²) / 2. Monitoring continued until day 21. After the experiment was terminated, all experimental animals were sacrificed, and the subcutaneous tumor tissue was completely dissected. The tumor was weighed immediately after dissection, and the data were recorded. The tumor mass (unit: g) was measured using an electronic balance to assess the effect of Aldoc gene knockdown on tumor burden.

[0077] The results are as follows Figures 4-6 As shown, the subcutaneous tumors in the control group mice grew rapidly, and the tumor volume and mass were significantly higher than those in the Aldoc-shRNA1 group, indicating that knocking down the Aldoc gene can effectively inhibit the in vivo tumorigenicity of breast cancer cells EO771.

[0078] Example 2: To further verify the applicability of the ALDOC knockdown method of this application in human breast cancer cells, human breast cancer cells MCF7 and BT549 were selected for knockdown experiments, and the knockdown effect and its impact on cell proliferation were evaluated. In this example, human ALDOC shRNA plasmids were used to transfect human breast cancer cells MCF7 and BT549 to construct a cell model with stable ALDOC knockdown. Transfection experiments were performed using two shRNA plasmids with different sequences, ALDOC-shRNA1 and ALDOC-shRNA2. After transfection, the mRNA expression level of the ALDOC gene was detected by RT-qPCR, and the ALDOC protein expression level was detected by Western blotting to evaluate the knockdown efficiency. This included synthesizing two shRNAs (ALDOC-shRNA1 and ALDOC-shRNA2) targeting the human ALDOC gene, cloning them into the pLKO.1 vector, and transfecting them into BT549 and MCF7 cells using Lipo8000 transfection reagent. Only the control plasmid shnc was transfected; cells without shRNA served as the control group. The specific steps included:

[0079] (1) Prepare 5×10⁵ cells / year of complete culture medium for human cancer cell lines, including human breast cancer cells MCF7 and BT549. 4 Cell suspension of cells / mL was added, and then 100 μL of the above-mentioned density of cell suspension was added to each well of a 96-well plate, for a total of 12 wells, with 3 wells serving as a relative control group. The cells were cultured at 37°C for 48 h until the cell confluence was 60%~70%.

[0080] (2) Take out the human ALDOC shRNA plasmid stored in a low-temperature environment and thaw it slowly on ice;

[0081] (3) Prepare the following mixture for each well: 100 ng ALDOC shRNA plasmid, 5 μL serum-free culture medium and 0.16 μL Lipo8000. After standing at room temperature for 15 min, add the mixture to the corresponding well. After 6 h of transfection, replace the medium with complete medium and continue culturing.

[0082] (4) Continue culturing for 2-3 days, during which time the nutrient medium can be supplemented or replaced to maintain cell viability;

[0083] (5) Cells were collected 48 h after transfection. RT-qPCR was used to observe the knockdown efficiency and Western Blot was used to verify changes in protein expression. The mRNA and protein expression were significantly inhibited, indicating that the cell line was successfully transfected.

[0084] The two nucleotide sequences of human ALDOC shRNA are shown below (both with pLKO.1 backbone):

[0085] ALDOC-shRNA1: 5′-GCTTTGAGTTCGATGTTAAAG-3′ (SEQ ID NO:7)

[0086] ALDOC-shRNA2: 5′-GAGCCTGTGATCAGCAATAAT-3′ (SEQ ID NO:8)

[0087] Each shRNA was synthesized and cloned into the pLKO.1 backbone plasmid vector for transfection of human breast cancer cell lines BT549 and MCF7, achieving effective knockdown of ALDOC gene expression.

[0088] To verify the knockdown effect, the experimental cells were divided into the following experimental groups:

[0089] (1) Relative control group (shnc): Human breast cancer cells MCF7 and BT549, which were transfected only with the control plasmid shnc and not transfected with any shRNA plasmid, served as the negative control group;

[0090] (2) ALDOC-shRNA1 group (ALDOC KD1): human breast cancer cells MCF7 and BT549 were transfected with pLKO.1 plasmid containing ALDOC-shRNA1 (SEQ ID NO:7);

[0091] (3) ALDOC-shRNA2 group (ALDOC KD2): human breast cancer cells MCF7 and BT549 were transfected with pLKO.1 plasmid containing ALDOC-shRNA2 (SEQ ID NO:8).

[0092] The RT-qPCR method was used to verify the knockdown effect of ALDOC in BT549 and MCF7 cells, including:

[0093] (1) Using RNA extraction reagent (Trizol), cells from the control group, ALDOC-shRNA1 group and ALDOC-shRNA2 group were collected, and total RNA was extracted and reverse transcription was performed.

[0094] (2) Real-time quantitative PCR, primer sequences:

[0095] ALDOC-Forward: 5′-TCTTCCCTCCCTCTCTC-3′ (SEQ ID NO:9)

[0096] ALDOC-Reverse: 5′-GACCCAGCCATCCTGTTC-3′ (SEQ ID NO:10)

[0097] ACTB-Forward: 5′-TCTCCCAAGTCCACACAGG-3′ (SEQ ID NO:11)

[0098] ACTB-Reverse: 5′-GGCACGAAGGCTCATCA-3′ (SEQ ID NO:12)

[0099] (3) Prepare the reagents according to Table 1, operate on ice, and mix well;

[0100] (4) Add the mixed liquid to the real-time PCR plate. Seal the real-time PCR plate with sealing film, protect it from light, and centrifuge briefly.

[0101] Ct was obtained using StepOne software, with ACTB as an intrinsic parameter; ΔCt = Ct(target) - Ct(ACTB), and the relative expression level = 2^-ΔΔCt.

[0102] The results are as follows Figure 7 and Figure 8 As shown, in BT549 and MCF7 cells transfected with ALDOC-shRNA1 (ALDOC KD1) and ALDOC-shRNA2 (ALDOCKD2), the expression levels of ALDOC mRNA and protein were significantly reduced compared to the control group (shnc), indicating that ALDOC knockdown could be successfully achieved in different human breast cancer cells, demonstrating that shRNA plasmids have a good interference effect on target genes. Among them, ALDOC-shRNA1 knockdown efficiency was higher; therefore, the cell line constructed by transfecting with ALDOC-shRNA1 was selected for subsequent experiments.

[0103] To further investigate the effect of ALDOC knockdown on tumor proliferation, MCF7 and BT549 cell lines transfected with the ALDOC-shRNA1 plasmid were used for CCK8 assays. The absorbance (OD450) values ​​were measured at 0h, 24h, 48h, 72h, and 96h post-transfection to assess cell viability. Specific steps included:

[0104] Logarithmic growth phase MCF7 and BT549 cells were digested and prepared into cell suspensions. After counting, they were seeded into 96-well plates at a density of 5 × 10³ cells per well, with 3 technical replicates per group. At five time points after cell attachment (0 h, 24 h, 48 h, 72 h, and 96 h), the complete culture medium in the experimental wells was removed, and 10 μL of CCK-8 working solution (CCK-8 reagent and basal culture medium were prepared at a ratio of 1:9) was added to each well. After incubation at 37°C for 2 h, the absorbance (OD450) was read at a wavelength of 450 nm. The OD values ​​were recorded at each time point, and the cell proliferation rate was calculated.

[0105] Experimental results are as follows Figure 9 As shown, in BT549 and MCF7 cells, the proliferation rates of ALDOC-shRNA1 and ALDOC-shRNA2 transfection groups were significantly lower than those of the control group at 48h (day 3), 72h (day 4), and 96h (day 5), with statistically significant differences (*P<0.05). The difference further widened at 96h (day 5), indicating that ALDOC knockdown can significantly inhibit the proliferation of breast cancer cells.

[0106] Example 3: Rosiglitazone treatment of tumor cells with knocked-down ALDOC expression

[0107] Based on Example 2, MCF7 and BT549 breast cancer cells with knocked-down ALDOC expression were treated in vitro with the PPARγ agonist rosiglitazone (Rosi) to evaluate the antiproliferative effect of the combined treatment.

[0108] Experimental methods:

[0109] (1) Cell lines (MCF7 cells and BT549 cells) transfected with ALDOC-shRNA1 plasmid were seeded in logarithmic growth phase at 5×10³ cells / well in 96-well plates, with 3 technical replicate wells in each group, and cultured overnight at 37°C in a 5% CO2 incubator.

[0110] (2) After the cells adhered, different concentrations of rosiglitazone (10 μM, 20 μM, 50 μM) were added. The stock solution was dissolved in DMSO and diluted in serum-free culture medium, with a final concentration not exceeding 0.1% DMSO. An equal volume of DMSO dilution was added to the control group.

[0111] (3) After incubating for 48 hours, add 10 μL of CCK-8 working solution (CCK-8 reagent and basal culture medium are prepared in a 1:9 ratio) to each well, incubate at 37°C for 2 hours, read the absorbance value (OD450) at 450 nm using an ELISA reader, and record the data for each group.

[0112] (4) Set up the following experimental groups respectively:

[0113] Relative control group (shnc+DMSO): Transfected with control plasmid and treated with the same concentration of DMSO as the experimental group;

[0114] ALDOC-shRNA1 group (ALDOC KD1): ALDOC-shRNA1 was transfected and treated with the same concentration of DMSO as the experimental group;

[0115] Rosi: Transfected with control plasmid and treated with different concentrations of rosiglitazone;

[0116] Combined treatment group (ALDOC KD1+Rosi): ALDOC-shRNA1 was transfected and treated with different concentrations of rosiglitazone.

[0117] (5) Calculate the cell proliferation inhibition rate according to the following formula:

[0118] Inhibition rate (%) = [1 - (OD of test wells - OD of blank wells) / (OD of control wells - OD of blank wells)] × 100%

[0119] The blank wells are those containing only culture medium and CCK-8.

[0120] (6) To evaluate the combined action of ALDOC knockdown and rosiglitazone, the synergistic effect of ALDOC knockdown and 10 μmol / L rosiglitazone was calculated.

[0121] The results are as follows Figure 9 , Figure 10 and Figure 11 As shown, in BT549 cells, ALDOC knockdown (25.2% inhibition rate) combined with 10 μM rosiglitazone (22.2% inhibition rate) achieved an inhibition rate of 65.6%, which was 18.2% higher than the theoretical summation value (47.4%) (p=0.003), and the Bliss co-inhibition score was 23.8%.

[0122] In MCF7 cells, the combined inhibition rate reached 70.4%, which was 23.0% higher than the theoretical summation value (p=0.002), and the Bliss co-inhibition score was 28.3%.

[0123] Table 2. Analysis of the antiproliferative effects and synergistic effects of ALDOC knockdown combined with rosiglitazone treatment in breast cancer cells.

[0124]

[0125] These results indicate that the combined action of the PPARγ agonist rosiglitazone and ALDOC expression inhibitor can significantly enhance the anti-breast cancer cell proliferation activity, providing a basis for subsequent in vivo validation.

[0126] Example 4: Therapeutic effect of rosiglitazone combined with ALDOC knockdown on mouse breast cancer xenografts

[0127] (1) Aldoc-shRNA1 in the logarithmic growth phase was transfected into EO771 cells and then digested to prepare 5×10 6 Cell suspension of 5 × 10⁶ cells / mL, take 100 μL of suspension (containing 5 × 10⁶ cells / mL). 5(1 cell) was injected subcutaneously into the right axilla of 6-8 week old female C57BL / 6 mice. Aldoc-shRNA1 transfection of EO771 cells was performed using the Aldoc-knockdown EO771 cell line constructed in Example 1, with the nucleotide sequence 5′-GCCAGTCTTGATCAGGACTTT-3′ (SEQ ID NO:1), and the vector was the pLKO.1 backbone.

[0128] (2) When the tumor volume reaches 50-100 mm³, the mice are randomly divided into eight groups (n=5 per group):

[0129] Control group (shNC+DMSO): Cells transfected with the control plasmid (not transfected with Aldoc-shRNA1) were inoculated only and injected intraperitoneally with PBS (100 μL) containing 5% DMSO daily.

[0130] Rosiglitazone group (shNC+Rosi): cells were inoculated with control plasmid transfected cells and simultaneously injected intraperitoneally daily with 10 mg / kg rosiglitazone (dissolved in PBS containing 5% DMSO).

[0131] Palmitic acid group (shNC+PA): Control cells were transfected with the control plasmid and pretreated with complete medium containing 100 μmol / L palmitic acid for 48 h before subcutaneous tumor formation to simulate metabolic stress in vitro. After pretreatment, the cells were digested and collected at a ratio of 5 × 10⁶ cells per mouse. 5 A dose of one cell was injected subcutaneously into the right axilla of mice;

[0132] Rosiglitazone-PA group (shNC+Rosi+PA): Before subcutaneous tumor formation, control cells (shNC) were pretreated with complete culture medium containing 100 μmol / L palmitic acid for 48 h to simulate metabolic stress in vitro. After pretreatment, the cells were digested and collected at a concentration of 5 × 10⁶ cells per mouse. 5 A dose of 1000 cells was injected subcutaneously into the right axilla of mice. After tumor formation, 10 mg / kg of rosiglitazone was injected intraperitoneally daily (solvent and administration regimen were the same as the rosiglitazone group).

[0133] Aldoc-shRNA1 group (Aldoc KD1+DMSO): cells were only inoculated with Aldoc-shRNA1 transfected cells, without any drug treatment;

[0134] Combined treatment group 1 (Aldoc KD1+Rosi): cells were inoculated with Aldoc-shRNA1 transfected cells and 10 mg / kg rosiglitazone was injected intraperitoneally daily;

[0135] Combined treatment group 2 (Aldoc KD1+PA): cells were inoculated with Aldoc-shRNA1 transfected cells and treated with 100 μmol / L palmitic acid, in the same manner as the rosiglitazone-PA group;

[0136] Combined treatment group 3 (Aldoc KD1+Rosi+PA): cells were inoculated with Aldoc-shRNA1 transfected cells and treated with 100 μmol / L palmitic acid, and rosiglitazone was injected intraperitoneally daily, in the same manner as the rosiglitazone-PA group.

[0137] (3) During the administration period, the long and short diameters of the tumor were measured every 2 days using vernier calipers. The tumor volume (unit: mm³) was calculated according to the formula V=(long × short²) / 2. The monitoring was carried out continuously for 21 days. The measurement was performed using a double-blind method, with two experimenters recording the data independently and taking the average value.

[0138] (4) After the experiment was terminated, all experimental animals were euthanized, the subcutaneous tumor tissue was completely removed, and the tumor mass was immediately weighed and recorded (unit: mg).

[0139] (5) The treatment effect was confirmed by comparing the tumor volume growth curves and final tumor weight of each group. The results showed that the tumor growth in the combined treatment group was significantly inhibited.

[0140] The results are as follows Figure 12 As shown, the tumor volume after treatment with shNC+DMSO was not significantly reduced compared to either the rosiglitazone group (shNC+Rosi) or the palmitic acid group (shNC+PA) alone. However, compared to either the rosiglitazone group or the palmitic acid group, the combined use of rosiglitazone and palmitic acid (rosiglitazone-PA group) significantly reduced the tumor volume, indicating that the combined use of rosiglitazone and palmitic acid is an effective measure to inhibit tumor growth. Furthermore, compared to the shNC+DMSO group, knocking down the Aldoc gene in the Aldoc-shRNA1 group significantly inhibited tumor development. Moreover, the Aldoc KD1+Rosi group showed even better tumor inhibition, demonstrating that the Aldoc gene knockdown combined with rosiglitazone effectively inhibited tumor growth in this application. Additionally, the combined treatment group 2 (Aldoc KD1+PA) showed that Aldoc gene knockdown combined with palmitic acid also effectively inhibited tumor growth, but its inhibitory effect was weaker than that in combined treatment group 1. The results from combination therapy group 3 (Aldoc KD1+Rosi+PA) show that knocking down the Aldoc gene in combination with palmitic acid and rosiglitazone can maximally inhibit tumor development.

[0141] Figure 12 The tumor suppression results of different treatment groups in the middle part are as follows: Figure 13 and Figure 14As shown, the tumor volume in the control group (shNC+DMSO) increased from 107±6 mm³ on day 7 to 1218±123 mm³ on day 21; in the Aldoc-shRNA1 group (AldocKD1+DMSO), it was 556±37 mm³ (inhibition of 54.4%); in the rosiglitazone group (shNC+Rosi), it was 879±81 mm³ (inhibition of 27.8%); and in the combination therapy group 1 (Aldoc KD1+Rosi), it was 297±27 mm³ (inhibition of 75.6%, p<0.01). Figure 13 As shown, the corresponding tumor weights were 1.099±0.063g, 0.667±0.035g, 1.027±0.086g, and 0.391±0.041g, respectively. The combined treatment group 1 was significantly better than the single treatment group (p<0.01). According to the Bliss model, the actual inhibition rate of the combined group on day 21 was 75.6%, higher than the predicted value of 67.9%, indicating a synergistic effect between the two.

[0142] In summary, in a mouse breast cancer model, the combined effect of Aldoc expression inhibition and rosiglitazone showed superior antitumor efficacy compared to either drug alone. This quantitative analysis further validated the in vitro experimental results of Example 3, indicating that Aldoc knockdown significantly enhances the antitumor effect of rosiglitazone, providing a theoretical basis for clinical combination therapy.

[0143] Furthermore, combining PA with other drugs can enhance the antitumor effects of rosiglitazone or Aldoc knockdown.

[0144] Example 5: Study on the anti-tumor effect mechanism of ALDOC

[0145] As can be seen from the above examples, knockdown of ALDOC can significantly enhance the antitumor effect of rosiglitazone. It is speculated that ALDOC can bind to HSP90 and compete with the binding of PPARγ on HSP90, thereby reducing the amount of PPARγ and HSP90, affecting the activity of the PPARγ signaling pathway and weakening its antitumor effect.

[0146] To verify the above mechanism hypothesis, this application embodiment uses an ALDOC overexpression system to detect its effect on the binding of PPARγ to HSP90. Specifically, the following steps are included:

[0147] The following expression plasmids were constructed in the embodiments of this application:

[0148] (1) FLAG-ALDOC plasmid: The human ALDOC coding sequence was cloned into the pcDNA3.1 vector with a FLAG tag at the N end;

[0149] (2) HA-HSP90 plasmid: The human HSP90 coding sequence was cloned into the pcDNA3.1 vector with an N-terminal HA tag;

[0150] (3) myc-PPARG plasmid: The human PPARG coding sequence was cloned into the pcDNA3.1 vector with a myc tag at the N end.

[0151] After confirmation by sequencing, each plasmid was transfected into MCF7 and BT549 breast cancer cells, respectively. The empty vector pcDNA3.1 transfection group served as a negative control. The transfection reagent was Lipo8000, and the following steps were performed:

[0152] Logarithmic growth phase MCF7 and BT549 cells were digested and resuspended in complete culture medium at a cell density of 5 × 10⁻⁶. 6 The plasmids were seeded at a density of 100 ng / mL into multi-well plates according to the experimental design. The FLAG-ALDOC, HA-HSP90, and myc-PPARG plasmids, stored at low temperatures, were removed and thawed on ice. A transfection mixture was prepared: 100 ng of each plasmid, 5 μL of serum-free medium, and 0.16 μL of Lipo8000 transfection reagent. After standing at room temperature for 15 min, the mixture was added to the wells. Six hours after transfection, the medium was replaced with complete medium, and the plates were incubated for another 48 hours.

[0153] (1) Experiment on the combination of FLAG-ALDOC and HSP90

[0154] In MCF7 and BT549 cell lines, overexpression plasmids containing FLAG-ALDOC were transfected, and immunoprecipitation experiments were performed using FLAG-conjugated magnetic beads.

[0155] 48 hours after transfection, cell lysates were collected and immunoprecipitated with anti-HSP90 antibody. 5% of the total protein lysate was used as the input sample for IB (immunoblotting) detection of the binding of HSP90 and FLAG-ALDOC.

[0156] In MCF7 and BT549 cell lines, transfection with HA-HSP90 plasmid was performed, and immunoprecipitation experiments were conducted using HA-coupled magnetic beads.

[0157] Forty-eight hours after transfection, cell lysates were collected and subjected to immunoprecipitation using anti-ALDOC antibody. 5% of the total protein lysate was used as the input sample for Western blotting (IB) detection of the binding of HSP90 to FLAG-ALDOC. The results are as follows: Figure 15 As shown, this suggests that the two can form a complex.

[0158] (2) Experiment on the binding of myc-PPARγ with HSP90

[0159] In MCF7 and BT549 cell lines, overexpression plasmids containing myc-PPARG were transfected, and myc-coupled magnetic beads were used for immunoprecipitation experiments.

[0160] Forty-eight hours after transfection, cell lysates were collected and immunoprecipitated using anti-HSP90 antibody. 5% of the total protein lysate was used as the input sample for Western blotting (IB) to detect the binding of HSP90 to myc-PPARγ. The results are as follows: Figure 16 As shown, the results suggest that the two can form a complex.

[0161] (3) Effect of ALDOC on the binding of PPARγ-HSP90

[0162] FLAG-ALDOC and myc-PPARG plasmids were transfected into MCF7 and BT549 cell lines, and immunoprecipitation was performed using FLAG-coupled magnetic beads and myc-coupled magnetic beads.

[0163] 48 hours after transfection, cell lysates were collected and immunoprecipitated using anti-HSP90 antibody and FLAG antibody. 5% of the total protein lysate was used as the input sample for IB detection of the binding of HSP90 and myc-PPARγ.

[0164] The results are as follows Figure 16 As shown in the results, under ALDOC overexpression conditions, the HSP90-PPARγ coprecipitation signal was significantly weakened, suggesting that ALDOC can weaken the binding of HSP90 and PPARγ.

[0165] Gradient transfection ALDOC validates competitive binding

[0166] A transfection gradient of FLAG-ALDOC plasmid was established (total DNA amount was kept consistent by supplementing with empty vector pcDNA3.1), and co-transfected with myc-PPARG and HA-HSP90 plasmids into MCF7 or BT549 cells. Specifically, 0 μg, 0.5 μg, 1.0 μg, and 2.0 μg of FLAG-ALDOC plasmid were added to each well (using a 6-well plate as an example), while 1.0 μg of myc-PPARG plasmid and 1.0 μg of HA-HSP90 plasmid were added simultaneously, and the total DNA amount in each well was supplemented with empty vector to the same level (4.0 μg). Co-transfection was performed using Lipo8000 transfection reagent, and the culture medium was replaced with complete medium after 6 hours of transfection for continued culturing.

[0167] Cell lysates were collected 48 h post-transfection. 5% of the total protein lysate was used as the input sample for co-immunoprecipitation experiments: Firstly, FLAG-conjugated magnetic beads were used for co-immunoprecipitation to enrich the FLAG-ALDOC complex, and Western blotting (IB) was performed to detect the binding of HSP90 to FLAG-ALDOC. Secondly, myc-conjugated magnetic beads were used for co-immunoprecipitation to enrich the myc-PPARγ complex, and IB was performed to detect the binding of HSP90 to myc-PPARγ. The competitive binding relationship between ALDOC and PPARγ to HSP90 was assessed by comparing the changes in co-precipitation signal intensity of HSP90 with FLAG-ALDOC and myc-PPARγ under different FLAG-ALDOC transfection gradients. The results are shown below. Figure 17 As shown.

[0168] The bound proteins were analyzed by SDS-PAGE and Western Blot, and were labeled and detected using antibodies against PPARγ, HSP90, FLAG, and myc.

[0169] from Figures 15-18 The results showed that in breast cancer cells exogenously transfected with both FLAG-ALDOC and HA-HSP90, ALDOC could bind to HSP90, such as... Figure 15 As shown. In breast cancer cells simultaneously transfected with myc-PPARγ and HA-HSP90, PPARγ can bind to HSP90, as... Figure 16 As shown. Transfection with FLAG-ALDOC and myc-PPARγ simultaneously or at different times revealed that in breast cancer cells transfected with FLAG-ALDOC, the binding degree of PPARγ and HSP90 was reduced, as shown in the figure. Figure 17 As shown in the figure. Next, the amount of FLAG-ALDOC plasmid transfected into breast cancer cells was increased according to a concentration gradient. It was found that in breast cancer cells with gradually increasing ALDOC expression, the binding degree of PPARγ and HSP90 gradually decreased, as shown in the figure. Figure 18 As shown in the figure, the results above suggest that ALDOC competes with PPARγ for binding to HSP90, and the gradual increase in ALDOC protein expression affects the protein expression level of PPARγ.

[0170] This application investigated the interactions between ALDOC, HSP90, and PPARγ by transfecting the MCF7 and BT549 breast cancer cell lines with myc-PPARγ, HA-HSP90, and FLAG-ALDOC plasmids, and by using co-immunoprecipitation and Western blotting. The competitive co-immunoprecipitation assay revealed how ALDOC affects the binding of PPARγ to HSP90 at different transfection levels. Experimental data showed that ALDOC overexpression significantly reduced the binding of PPARγ to HSP90 in a dose-dependent manner, suggesting that ALDOC may affect PPARγ function by competitively binding to HSP90. However, the implementation of this application does not depend on this mechanism and does not constitute a limitation on the scope of protection of this application.

[0171] Statistical analysis methods

[0172] SPSS 19.0 software was used for statistical analysis. All continuous data were first tested for normality and expressed as mean ± standard deviation (SD). For normally or approximately normally distributed continuous data, one-way ANOVA was used for analysis of differences among multiple groups, and LSD-t tests were used for pairwise comparisons between groups. Data visualization was performed using GraphPad Prism 9.0 software to create scatter plots, bar charts, line graphs, etc., and standard error bars were used to illustrate data variability.

[0173] The above description is merely an embodiment of this application and does not limit the patent scope of this invention. Any equivalent structural transformations made using the content of this application's specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of this invention.

[0174] In the above embodiments, the descriptions of each embodiment have different focuses. For parts not described in detail in a certain embodiment, please refer to the relevant descriptions in other embodiments.

[0175] The above provides a detailed description of an antitumor preparation and its application provided by the embodiments of this application. Specific examples have been used to illustrate the principles and implementation methods of this application. The description of the above embodiments is only for the purpose of helping to understand the method and core ideas of this application. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the ideas of this application. Therefore, the content of this specification should not be construed as a limitation of this application.

Claims

1. An antitumor agent, characterized in that, Include: (a) Rosiglitazone; (b) An inhibitor for reducing the expression of fructose-1,6-bisphosphate aldolase C; wherein the inhibitor for reducing the expression of fructose-1,6-bisphosphate aldolase C is a shRNA targeting the fructose-1,6-bisphosphate aldolase C mRNA sequence, wherein the shRNA sequence is as shown in SEQ ID NO:1 or SEQ ID NO:

7.

2. The antitumor agent according to claim 1, characterized in that, The shRNA sequence was constructed in a plasmid vector.

3. The antitumor agent according to claim 2, characterized in that, The skeletal structure of the plasmid vector is the pLKO.1 vector.

4. The antitumor agent according to claim 1, characterized in that, The antitumor agent also contains palmitic acid.

5. The antitumor agent according to any one of claims 1 to 4, characterized in that, The antitumor preparation also contains a pharmaceutically or immunologically acceptable diluent.

6. The antitumor agent according to any one of claims 1 to 4, characterized in that, The antitumor preparation also contains pharmaceutically or immunologically acceptable excipients.

7. The antitumor agent according to any one of claims 1 to 4, characterized in that, The antitumor agent also contains a pharmaceutically or immunologically acceptable carrier.

8. The use of an antitumor agent as described in any one of claims 1 to 4 in the preparation of an antitumor drug, wherein the tumor is breast cancer.

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