Product for reducing phosphorylation level of mitochondrial key apoptosis protein BAD protein serine 99 site and anti-tumor application thereof

Abstract

The invention discloses a product for reducing the phosphorylation level of a Serine 99 site of a BAD protein and an anti-tumor application of the product. The invention provides the BAD S99A mutant, phosphorylation is specifically blocked through the BAD S99A mutant, accurate regulation and control of an apoptosis pathway are achieved, and the problems that in the prior art, due to the fact that the phosphorylation state of BAD cannot be controlled, the apoptosis activation efficiency is low, and the mechanism is indefinite are solved.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to products that reduce the phosphorylation level of serine 99 site of BAD protein, a key apoptosis protein in mitochondria, and their anti-tumor applications. Background Technology

[0002] The BAD (Bcl-2 Antagonist of Cell Death) gene is a member of the Bcl-2 family and encodes a cell death-related protein. This gene plays a crucial role in the regulation of apoptosis, primarily regulating the mitochondrial-mediated apoptosis pathway through its protein product, BAD. When unphosphorylated, BAD binds to anti-apoptotic proteins in the Bcl-2 family (such as Bcl-2 and Bcl-xL), thereby inhibiting their function and promoting apoptosis. Conversely, upon phosphorylation by specific kinases, BAD's affinity for anti-apoptotic proteins decreases, leading to their release and further activating the apoptosis pathway by promoting the activation of pro-apoptotic factors such as Bax and Bak. The phosphorylation regulation of BAD is mediated by several key kinases (such as Akt, PKA, AMPK, JNK, and GSK-3β), and the abnormally high expression or activity of these kinases is often the basis for the development and drug resistance of many cancers. Akt inhibits the binding of BAD to Bcl-2 or Bcl-xL by phosphorylating the Ser99 site of BAD, thereby weakening the inhibitory effect of BAD against apoptosis proteins and promoting cell survival. Akt activation is usually initiated through the PI3K (phosphatidylinositol 3-kinase) pathway and is a key regulator of cell proliferation, survival, and metabolism. Akt upregulation is often closely related to the occurrence and progression of cancer, especially showing high expression in various malignant tumors such as breast cancer, prostate cancer, non-small cell lung cancer, gastric cancer, and ovarian cancer. PKA is activated through the cAMP signaling pathway and can phosphorylate the Ser75 site of BAD, thereby inhibiting the pro-apoptotic function of BAD. The role of PKA activation is usually related to cell survival and metabolism, especially in tumors of the endocrine system (such as adrenal tumors and ovarian cancer), where abnormal expression of PKA affects the regulation of BAD, thereby altering the apoptosis threshold. AMPK mainly regulates cellular energy metabolism and modulates its activity by phosphorylating the Ser99 site of BAD. Under nutrient deficiency or hypoxic conditions, AMPK activation helps cells survive under stress. Aberrant activation or inhibition of AMPK is often associated with tumor metabolic reprogramming and has shown importance in certain colorectal cancers, pancreatic cancers, and liver cancers. JNK is a key regulator of stress response; it can enhance cellular apoptosis by phosphorylating the Ser128 site of BAD, prompting BAD to release the anti-apoptotic protein Bcl-2. JNK activation is generally associated with cellular responses to oxidative stress or DNA damage; in liver cancer, gastric cancer, and lung cancer, high JNK expression is associated with poor prognosis. GSK-3β usually inhibits the pro-apoptotic function of BAD by phosphorylating the Ser112 site of BAD. High expression or activation of GSK-3β can promote cell survival and proliferation, and its high expression in various cancer types (such as breast cancer, liver cancer, and pancreatic cancer) is closely related to the cell's ability to resist apoptosis.

[0003] Targeted therapy is an emerging cancer treatment strategy. Its core concept is based on a deep understanding of the mechanisms of tumor development and progression, selectively intervening in molecular targets specific to tumor cells to achieve precise killing of tumor cells while minimizing damage to normal cells. Compared to traditional chemotherapy drugs, targeted therapy has advantages such as fewer side effects, more precise treatment effects, and a higher degree of personalization, and has been widely used in the treatment of various solid tumors, including breast cancer, lung cancer, and gastric cancer. In the field of ovarian cancer, although some targeted drugs, including PARP inhibitors and VEGF inhibitors, have been approved for clinical use, effective targeted interventions are still lacking for the treatment of cisplatin-resistant (CR) ovarian cancer.

[0004] In the field of tumor biology, the study "Construction of Eukaryotic Expression Plasmids for Bax and Bad Genes and Observation of Translational Levels after Transfection into Human Embryonic Kidney Cells" successfully constructed eukaryotic expression plasmids pcDNA3.1-Bax and pcDNA3.1-Bad for Bcl-2-related X protein (Bax) and Bcl-2-related cell death factor (Bad), respectively, and achieved high-efficiency expression in HEK293 cells. This study amplified the cDNA sequences of Bax and Bad using RT-PCR and cloned them into the pcDNA3.1 vector, subsequently introducing them into HEK293 cells via liposome transfection. Western blotting results confirmed the successful expression of Bax and Bad proteins. However, this study only transfected the complete genome sequence of Bad and did not specifically regulate its phosphorylation state; therefore, it was impossible to investigate the role of phosphorylation in apoptosis. The study, titled "Construction of Lentiviral Expression Vectors Carrying Human AKT2, PDK1, and BAD Genes and Their Expression in 293T Cells," successfully constructed a green fluorescent lentiviral vector carrying the human serine / threonine protein kinase B (AKT2), ​​phosphoinositol-dependent kinase-1 (PDK1), and bcl-2-related death protein (BAD) genes, and achieved high-efficiency expression in 293T cells. The AKT2, PDK1, and BAD genes in NSCLC tissues were amplified by RT-PCR, and the corresponding lentiviral expression vectors were constructed and successfully transfected into 293T cells. Western blot analysis showed the expression of AKT2, BAD, and PDK1 proteins in 293T cells. However, this study only used the lentiviral vector to transfect the complete genome sequence of BAD and did not specifically regulate the phosphorylation status of BAD, thus preventing the study of its phosphorylation function.

[0005] However, there are currently no targeted drugs specifically targeting BAD phosphorylation in clinical use, and there is a lack of mature drug molecules to effectively intervene in the phosphorylation state of this site. Therefore, developing novel targeted small molecule inhibitors or gene tools can not only restore the pro-apoptotic function of BAD, but also serve as an innovative targeted therapy strategy to reverse the resistance of ovarian cancer cells to cisplatin, which has significant research value and clinical application prospects. Summary of the Invention

[0006] The purpose of this invention is to address the above-mentioned shortcomings of the prior art by providing a mutated BAD protein and its encoding gene.

[0007] Another objective of this invention is to provide a plasmid-engineered bacterium that reduces the phosphorylation level of the BAD protein at the Serine 99 site.

[0008] Another object of the present invention is to provide the application of a reagent for reducing the phosphorylation level of the BAD protein at Serine 99 site.

[0009] A fourth objective of this invention is to provide a cisplatin-based treatment regimen that reduces phosphorylation levels at the Serine 99 site of the BAD protein.

[0010] The objective of this invention can be achieved through the following technical solutions:

[0011] A mutant BAD protein in which the 99th amino acid residue is mutated from serine in the wild type to alanine.

[0012] As a preferred embodiment of the present invention, the amino acid sequence of the BAD protein is shown in SEQ ID NO.1.

[0013] The gene encoding the mutated BAD protein described herein has the nucleotide sequence shown in SEQ ID NO.2.

[0014] A plasmid that reduces the phosphorylation level of the BAD protein at Serine 99 site contains a gene encoding the mutant BAD protein, preferably the gene shown in SEQ ID NO.2.

[0015] As a preferred embodiment of the present invention, the plasmid is obtained by inserting the gene encoding the mutated BAD protein into the BamHI and EcoR1 restriction sites of the pcDNA3.0 plasmid using pcDNA3.0 as the starting plasmid.

[0016] Genetically engineered bacteria containing the gene encoding the mutated BAD protein or the recombinant plasmid.

[0017] The application of reagents that reduce the phosphorylation level of Serine 99 in BAD protein in the preparation of antitumor drugs or drugs that improve the sensitivity of tumor cells to chemotherapy and / or radiotherapy, wherein the reagents that reduce the phosphorylation level of Serine 99 in BAD protein are reagents that mutate serine at position 99 of BAD protein to alanine.

[0018] The reagent used to reduce the phosphorylation level of BAD protein at Serine 99 site is selected from any one of the following: (I) the mutant BAD protein described in this invention;

[0019] (II) The gene described in this invention;

[0020] (III) The recombinant plasmids described in this invention;

[0021] (IV) The genetically engineered bacteria of the present invention;

[0022] As a preferred embodiment of the present invention, the reagent that reduces the phosphorylation level of BAD protein at Serine 99 site maintains the pro-apoptotic activity of BAD protein and inhibits the proliferation, clonogenic ability and self-renewal ability of tumor cells by reducing the phosphorylation level of BAD protein at Serine 99 site.

[0023] A composition for treating tumors, said composition being selected from any one of the following:

[0024] (I) The recombinant plasmid + chemotherapy drug described in this invention;

[0025] (II) The genetically engineered bacteria and chemotherapy drugs described in this invention;

[0026] (III) The compound treatment regimen described in this invention plus chemotherapy drugs;

[0027] The preferred chemotherapy drugs are cisplatin, carboplatin, and paclitaxel.

[0028] Beneficial effects:

[0029] This invention provides a multifunctional cancer treatment strategy combining gene mutation technology and small molecule inhibitor intervention, focusing on targeting the phosphorylation of Serine 99 on the BAD protein to overcome chemotherapy resistance. This invention mutates the 99th serine (Ser99) of the BAD protein to alanine (Ala), irreversibly blocking Ser99 phosphorylation by the absence of a hydroxyl group in the alanine side chain, mimicking the continuous dephosphorylation activity state of BAD. By constructing a pcDNA3.0 plasmid vector and introducing a specific S99 mutation, BAD phosphorylation is effectively inhibited, significantly inducing cancer cell apoptosis and demonstrating broad anti-tumor potential. This method has been validated in ovarian cancer cells and has the potential for widespread application in various malignant tumors, including lung cancer, ovarian cancer, breast cancer, and liver cancer, for personalized treatment of chemotherapy-resistant tumors. When used in combination with radiotherapy / chemotherapy, it can reverse tumor resistance by enhancing apoptosis sensitivity (e.g., by inhibiting the anti-apoptotic proteins BCL-2 / BCL-XL).

[0030] In addition, this invention screened and optimized a highly efficient small molecule inhibitor to inhibit the phosphorylation of BAD protein Ser99, and combined it with cisplatin to significantly enhance the sensitivity of cisplatin to drug-resistant EOC cells, reverse the drug resistance phenotype, and inhibit tumor cell survival, activity and 3D tumor organoid growth.

[0031] This invention systematically verified the inhibitory effect of small molecule inhibitors on BAD S99 phosphorylation using high-throughput transcriptome sequencing, real-time quantitative PCR (qPCR), Western blotting, cell viability analysis, and apoptosis detection. It confirmed that these inhibitors significantly improve the sensitivity of cisplatin-resistant ovarian cancer cells to chemotherapeutic drugs, induce apoptosis, and inhibit cell proliferation, migration, and invasion. Furthermore, this invention reveals that small molecule inhibitors simultaneously intervene in the PI3K / AKT and JAK / STAT signaling pathways, synergistically regulating the expression of apoptosis-related genes, thereby achieving anti-tumor effects and reversing drug resistance. Attached Figure Description

[0032] Figure 1 Map of pcDNA3.0 plasmid

[0033] Figure 2 The red area represents the inserted BAD. S99A Encoded sequence

[0034] Figure 3 : Figure 2 Alignment of the red region sequence Query with the wild-type BAD coding sequence Sbjct

[0035] Figure 4 Methods for establishing OVCAR-3CR resistant cell lines

[0036] Figure 5 : Schematic diagram of experimental design and bioinformatics analysis workflow. OVCAR-3 and OVCAR-3CR cells were treated with cisplatin, and RNA was extracted and sequenced. Bioinformatics analysis included data quality control, differential gene expression analysis, pathway enrichment analysis, and visualization of key signaling pathways to explore the mechanisms of chemotherapy resistance and the role of BAD-related pathways.

[0037] Figure 6 Experiment on spheroid formation efficiency of EOC cells transfected with WT or BADS99A plasmid after culturing in non-adherent culture dishes for 6-7 days.

[0038] Figure 7 Small molecule inhibitors inhibit BAD phosphorylation in both drug-resistant and drug-sensitive strains.

[0039] Figure 8 Small molecule enantiomers inhibit the proliferation of cisplatin-sensitive and drug-resistant EOC cells by suppressing phosphorylation at the BAD Ser99 site.

[0040] Figure 9 Small molecule enantiomers inhibit the self-renewal of cisplatin-sensitive and drug-resistant EOC cells by suppressing phosphorylation at the BAD Ser99 site.

[0041] Figure 10 Small molecule enantiomers inhibit the in vitro growth of cisplatin-sensitive and drug-resistant EOC cells by suppressing phosphorylation at the BAD Ser99 site.

[0042] Figure 11 Small molecule enantiomers inhibit the migration and invasion of cisplatin-sensitive and drug-resistant EOC cells by suppressing phosphorylation at the BAD Ser99 site.

[0043] Figure 12 The combination of small molecule inhibitors and cisplatin reduced the survival rate, cell viability, and three-dimensional growth of ovarian cancer cells in Matrigel.

[0044] Figure 13 Transfection and Small Molecule Inhibitor Process

[0045] Figure 14 Correlation analysis of KDG in EOC cells after treatment with NCK(S / R) and its S and R isoforms, and after transfection with S99A.

[0046] Figure 15 Analysis of key differentially expressed gene upregulation pathways after EOC transfection with S99A vector

[0047] Figure 16 Analysis of key differentially expressed gene downregulation pathways after EOC transfection with S99A vector Detailed Implementation

[0048] Example 1: Construction of expression vector

[0049] 1.1 Plasmids and Cells

[0050] pcDNA3.0 was used as the empty vector plasmid. The cells used in this example included human ovarian cancer epithelial cells A2780 and primary cultured human ascites-derived ovarian cancer cell line (AFC), cultured in DMEM (Gibco, USA) containing 10% serum; the culture medium contained 10 μg / mL streptomycin and 100 U / mL penicillin, and all cells were cultured in a constant temperature incubator at 37°C and 5% CO2. The pcDNA3.0 plasmid map is shown below. Figure 1 .

[0051] 1.2 Reagents

[0052] The PCR primers used in this embodiment were synthesized by Sangon Biotech (Shanghai) Co., Ltd.; PCR PremixTaq TM All reagents were purchased from TaKaRa (Japan); total RNA extraction kit was purchased from Beyotime Biotechnology Co., Ltd.; plasmid miniprep kit and gel purification kit were purchased from Aisjin Biotechnology (Hangzhou) Co., Ltd.; blood / cell / tissue genomic DNA extraction kit and competent Escherichia coli DH5α were purchased from Beyotime Biotechnology Co., Ltd.; restriction endonucleases BamHI and EcoR1 were purchased from TaKaRa (Japan); plasmid transfection reagent Lipofectamine™ 3000 was purchased from Invitrogen (USA).

[0053] 1.3 Methods

[0054] 1.3.1 Extraction of total RNA from cells

[0055] Total RNA was extracted from human ovarian epithelial cells A2780. First, A2780 cells with 80% confluence were selected, digested with trypsin, and then digestion was terminated with complete DMEM containing 10% serum. The cells were centrifuged at 500 rpm for 3 min at room temperature, and the supernatant was discarded. The cells were resuspended in PBS, centrifuged again, and the supernatant was discarded. Total RNA was extracted from human ovarian epithelial cells A2780 according to the instructions of the total RNA extraction kit.

[0056] 1.3.2 RNA reverse transcription into cDNA

[0057] RNA was reverse transcribed into cDNA according to the Thermo Fisher High-Capacity cDNA Reverse Transcription Kit instructions. 1.3.3 Multi-step PCR amplification of the human point mutation BADS99A sequence: PCR amplification primers were designed based on the human BAD gene coding sequence in the NCBI Genbank database.

[0058] PCR amplification primer 1:

[0059] 5′-caagcttggtaccgagctcgatgttccagatcccagagtttgagccgagt-3′ (The italicized part is the sequence designed by homologous recombination, SEQ ID NO.3);

[0060] PCR amplification primer 2:

[0061] 5′-aggttggggggcgc ccg gcgcgagcggccccga-3′ (The underlined part is the S99A mutation site, SEQ ID NO.4);

[0062] PCR amplification primer 3:

[0063] 5′-tcggggccgctcgcgc cgg gcgccccccaacct-3′ (the underlined part is the S99A mutation site, SEQ ID NO.5);

[0064] PCR amplification primer 4:

[0065] 5′- (The italicized part of the English text is the sequence designed by homologous recombination, SEQ ID NO.6).

[0066] Using the cDNA obtained in the previous step as a template, primers 1 and 2 were used to amplify the upstream BAD sequence containing the S99A mutation site; primers 3 and 4 were used to amplify the downstream BAD sequence containing the S99A mutation site; using the upstream and downstream BAD sequences containing the S99A mutation site as templates, primers 1 and 4 were used to amplify the full-length BAD sequence containing the S99A mutation site. Using the artificially synthesized human BAD gene from the NCBI Genbank database as a template, primers 1 and 4 were used to amplify the full-length wild-type BAD WT sequence.

[0067] The amplification system for the PCR reaction is as follows:

[0068]

[0069] *: The recommended amount of template DNA in a 50μL PCR reaction system is 0.1-1μg of human genome.

[0070] Premix Taq (Ex Taq Version 2.0plus dye) solution composition:

[0071] TaKaRa Ex Taq 1.25U / 25μL

[0072] dNTP Mixture 2×conc. 0.4mM each

[0073] Ex Taq Buffer 2×conc. 4mM Mg 2+

[0074] The PCR amplification procedure is as follows:

[0075]

[0076]

[0077] Each PCR amplification product was subjected to electrophoresis on a 1% agarose gel. After the PCR product band size was correctly identified, the product was excised and purified.

[0078] 1.3.4pcDNA3.0-BAD S99A Construction and identification of plasmids

[0079] (1) Recombination reaction

[0080] The pcDNA3.0 plasmid was double-digested with BamHI and EcoR1 at 37°C for 0.5 h. The linearized pcDNA3.0 plasmid was then recombined with the gel-recovered product.

[0081] The system for the recombination reaction is as follows:

[0082] 5×SE Cloning Buffer 2μL

[0083] Linearization vector 10-50 ng 2 μL (20 ng recommended for below 3 kb, 40 ng recommended for 6 kb, and 60 ng recommended for 9 kb)

[0084] Insert 5 μL fragment (X:Y1 molar ratio = 1:2 to 1:5)

[0085] SE Recombinase 1μL

[0086] Add ddH2O to bring the volume to 10 μL.

[0087] The conditions for the recombination reaction are: 37℃ for 30 min, then transfer to ice or store at -20℃.

[0088] (2) Transformation of competent E. coli cells with recombinant plasmids

[0089] Remove competent cells from the -80°C freezer and thaw them on ice. If aliquoting is required, transfer the freshly thawed cell suspension into sterile, pre-chilled centrifuge tubes and place them in an ice bath. The recommended volume for transforming E. coli competent cells is 100 μL, which can be aliquoted as needed. Note that the volume of DNA used should not exceed one-tenth of the competent cell suspension volume. The following experiment uses 100 μL of competent E. coli cells as an example.

[0090] Add the target DNA (i.e., the recombinant plasmid mentioned above) (10 ng, volume <10 μL) to the E. coli competent cell suspension, gently rotate the centrifuge tube to mix the contents, and incubate on ice for 30 minutes. Place the centrifuge tube in a 42°C water bath for 90 seconds, then quickly transfer it to ice for 3 minutes. (Note: Do not shake the centrifuge tube during this process). Add 900 μL of sterile LB medium (antibiotic-free) to the centrifuge tube, mix well, and incubate at 37°C with shaking for 1 hour (200 rpm). The purpose of this step is to induce the expression of the relevant resistance marker gene on the plasmid, thereby reviving the bacteria.

[0091] After centrifuging the transformed competent cells at low speed (5000 rpm, 4 minutes), discard some of the supernatant, retaining 100-150 μL of culture medium. Gently pipette the suspended cells and add them all to LB solid agar medium containing the appropriate antibiotic. Spread the cells evenly using a sterile bent-tip spreader. If the expected number of single colonies is large, the centrifugation step can be omitted, and 200 μL of transformation product can be directly spread onto a plate (spreading too much bacterial solution may cause single colonies to stick together and be difficult to pick). The remaining bacterial solution after spreading can be stored at 4°C. If the number of transformed colonies is too low the next day, the remaining bacterial solution can be spread onto a new plate.

[0092] Place the plate at room temperature until the liquid is absorbed, then invert the plate and incubate at 37°C for 12-16 hours to produce single colonies. The next day, randomly select single colonies and culture them in LB medium with shaking, amplify them in large quantities, and extract plasmids.

[0093] (3) pcDNA3.0-BAD S99A plasmid identification

[0094] The extracted recombinant plasmid was identified by double digestion with BamHI and EcoR1, and the sample was sent to Sangon Biotech (Shanghai) Co., Ltd. for nucleic acid sequencing. The sequencing results were compared using the software Snap Gene Viewer.

[0095] The recombinant plasmid sequencing alignment results showed that the inserted BAD S99A ( Figure 2 , Figure 3 The sequence is consistent with the theoretical sequence, indicating that the recombinant plasmid was successfully constructed.

[0096] Example 2. Introducing plasmids to achieve functional verification

[0097] In this invention, serine (Ser) contains a hydroxyl group (-OH), which is a key position for phosphorylation. To simulate the dephosphorylated state of the BAD protein, serine is mutated to alanine (Ala). Alanine's side chain consists only of a methyl group (-CH3) and lacks a hydroxyl group, thus preventing phosphorylation. Compared to serine, alanine is smaller and does not carry a negative charge, making it structurally and charge-wise similar to dephosphorylated serine. By mutating serine to alanine, the biological characteristics of the BAD protein in its dephosphorylated state can be effectively simulated, providing a foundation for further research on BAD function. Figure 4 ).

[0098] The plasmid was transiently transfected into cisplatin-sensitive OVCAR-3 and cisplatin-resistant OVCAR-3CR cells. Western blot analysis was used to verify transfection efficiency and phosphorylation regulation. Results showed that the pBADS99 / BAD ratio was significantly decreased in the BADS99A transfection group, indicating that the mutation successfully inhibited phosphorylation at the Ser99 site and maintained the pro-apoptotic activity of the BAD protein.

[0099] This change significantly inhibited cell proliferation, colony formation, and cell self-renewal capacity. Simultaneously, the S99A cell line exhibited lower tumorigenic potential, indicating the important role of BAD Ser99 phosphorylation in the proliferation, colony formation, stemness maintenance, and tumorigenesis of ovarian cancer cells. Figure 5-6 ).

[0100] Example 3. Small molecule compound intervention

[0101] 3.1 Compound Preparation

[0102] The small molecule inhibitor used was 2-((4-(2,3-dichlorophenyl)piperazin-1-yl)(pyridin-3-yl)methyl)phenol, abbreviated as NCK, including its R-enantiomer (dextral isomer), S-enantiomer (levorotatory isomer), and racemic mixture (NCK). All monomers and mixtures were obtained by chiral resolution and purification. The structural formulas are shown below:

[0103]

[0104] 3.2 Cell Culture

[0105] Human ovarian cancer cell line OVCAR-3 and its cisplatin-resistant substrain OVCAR-3CR were maintained in RPMI-1640 complete medium containing a mixture of 20% fetal bovine serum (FBS) and 1% penicillin-streptomycin and placed in a 37°C, 5% CO2 incubator.

[0106] 3.3 Compound Treatment

[0107] The R-isomer, S-isomer, and their mixture (NCK) were dissolved in DMSO and diluted to a working concentration of 2.5 μM. These solutions were then added to culture plates to treat cells for 12–18 hours. An equal volume of DMSO was added to the control group as a negative control.

[0108] 3.4 Protein Expression Detection (Western Blot)

[0109] After cell processing, lysed cells were collected, total protein was extracted, separated by SDS-PAGE, and transferred to a membrane. Immunoblot analysis was performed using anti-pBADS99 and anti-BAD antibodies. The phosphorylation level of BAD Ser99 was assessed by the pBADS99 / BAD ratio. Results showed that the R-isomer, S-isomer, and mixture of them (NCK) could inhibit BAD phosphorylation in both cisplatin-sensitive and cisplatin-resistant strains. Figure 7 ).

[0110] 3.5 Cell viability assay

[0111] OVCAR-3 and OVCAR-3CR cells were seeded at a density of 2000 cells / well in 96-well plates. After cell adhesion was stable, R-isomers, S-isomers, or mixtures thereof (NCK) were added for treatment. After 12–18 hours of treatment, Alamar Blue reagent (final concentration 10%) was added to each well, and the cells were incubated at 37°C for 3–4 hours. Fluorescence signals (excitation wavelength 490 nm, emission wavelength 590 nm) were detected using a multi-mode microplate reader to reflect cellular metabolic activity and thus assess cell viability. After treatment, cells were fixed with trichloroacetic acid (TCA), stained with 0.4% SRB for 30 minutes, and then rinsed several times with 1% acetic acid to remove background dye. After drying, Tris buffer (pH 10.5) was added to elute the dye, and absorbance was read at 560 nm. The SRB method uses protein content as an indirect indicator to reflect total cell count and proliferation capacity. The above test results show that the R-isomer, S-isomer, and their mixture (NCK) can all effectively inhibit the cell viability and proliferation of OVCAR-3 (cisplatin-sensitive strain) and OVCAR-3CR (cisplatin-resistant strain). Figure 8 Furthermore, these small molecule compounds exhibited significant inhibitory effects in both cell lines, suggesting that they have potential therapeutic value in inhibiting the growth of epithelial ovarian cancer cells.

[0112] 3.6 Sphere Formation Experiment

[0113] OVCAR-3 (cisplatin-sensitive) and OVCAR-3CR (cisplatin-resistant) cells were seeded at a density of 500 cells per well in 96-well plates with ultra-low adhesion, using RPMI medium containing 1×B27, 20 ng / mL EGF, and 20 ng / mL bFGF. After adding the R-isomer, S-isomer, or a mixture thereof (NCK), the cells were incubated at 37°C for 5–7 days. After culture, images were taken under a microscope, and the number and average diameter of spheroids formed were counted. The results showed that R, S, and NCK treatments significantly inhibited spheroid formation, suggesting that these compounds can effectively weaken the self-renewal capacity of cisplatin-sensitive and resistant EOC cells. Figure 9 ).

[0114] 3.7 Three-dimensional Matrigel in vitro culture model

[0115] Pre-cooled Matrigel matrix was evenly spread at the bottom of 24-well plates. After gel formation, 500–1000 EOC cells (OVCAR-3 or OVCAR-3CR) were suspended in medium containing small molecule inhibitors (R-isomer, S-isomer, or NCK) and added to the wells, allowing the cells to embed in the Matrigel. The cells were cultured at 37°C for 7–10 days, with the drug-containing medium being changed periodically. After culture, images were taken under a microscope, and the number and volume of cell colonies were counted to assess the effect of different treatments on the growth capacity of tumor organoids. Results showed that the small molecule inhibitors significantly inhibited the three-dimensional growth capacity of both cisplatin-sensitive and drug-resistant strains. Figure 10 ).

[0116] 3.8 Cell Migration and Invasion Assays Migration Assay: Treated OVCAR-3 and OVCAR-3CR cells (R-isomer, S-isomer, or NCK-treated) were suspended in serum-free medium and seeded in the upper chamber (8 μm pore size) of a Matrigel-free Transwell chamber. The lower chamber was supplemented with complete medium containing 10% FBS as a chemokine. After 24 hours of culture, unmigrated cells from the upper chamber were removed, fixed with methanol, and stained with crystal violet. Migrating cells from the lower chamber were counted under a microscope.

[0117] Invasion assay: The upper chamber of the Transwell was pre-coated with Matrigel (simulating the basement membrane), and the remaining steps were the same as the migration assay, but the incubation time was extended to 48 hours. After treatment, the number of invading cells that crossed the Matrigel matrix was counted by staining.

[0118] Experimental results showed that small molecule inhibitors could significantly inhibit the migration and invasion of cisplatin-sensitive and drug-resistant EOC cells, suggesting their potential role in inhibiting tumor invasion and metastasis. Figure 11 ).

[0119] 3.9 Combined Drug Trial

[0120] OVCAR-3 (cisplatin-sensitive) and OVCAR-3CR (cisplatin-resistant) cells were seeded in 96-well and 24-well plates, respectively. Prior to treatment, cells were cultured to the logarithmic growth phase in RPMI medium containing 2% fetal bovine serum. Cells were divided into three groups, and the treatment for each group was as follows:

[0121] Cisplatin alone (1 μM): 1 μM cisplatin was added to the cell culture medium and cultured for 18 hours. NCK alone (2.5 μM): 2.5 μM NCK was added to the cell culture medium and cultured for 18 hours. Cisplatin + NCK combined group: 1 μM cisplatin and 2.5 μM NCK were added to the cell culture medium and cultured for 18 hours.

[0122] Experimental procedure:

[0123] Alamar Blue assay: After 18 hours of treatment, add Alamar Blue dye to each well and incubate at 37°C for 3-4 hours. Use a microplate reader to measure fluorescence signals at 490 nm (excitation light) and 590 nm (emission light) wavelengths to assess cell viability and proliferation.

[0124] SRB assay (Sulforhodamine B assay): After 18 hours of treatment, cells were fixed with 20% ice-cold trichloroacetic acid for 1 hour, followed by staining with SRB dye for 30 minutes. After staining, excess dye was washed away with 1% acetic acid and the cells were air-dried. The absorbance was read at 560 nm using a microplate reader to assess the total protein content of the cells, indirectly reflecting cell proliferation and survival.

[0125] 3D Matrigel culture assay: Cells were seeded into pre-chilled Matrigel in 24-well plates (500–1000 cells per well). After the Matrigel solidified, culture medium containing small molecule inhibitors was added. Each group of cells was cultured for 7–10 days, during which the culture medium was changed and photographs were taken. Finally, the number and volume of cell spheroids were observed and evaluated under a microscope to further assess the cells' self-renewal capacity and tumor growth potential.

[0126] The above results indicate that the combination therapy of cisplatin and NCK showed a significant synergistic effect in inhibiting EOC cell proliferation, survival, and tumor growth. Figure 12Especially in cisplatin-resistant cells (OVCAR-3CR), combination therapy can effectively overcome cisplatin resistance and enhance treatment efficacy, suggesting the potential of this combination therapy strategy in the clinical treatment of cisplatin-resistant ovarian cancer.

[0127] Example 4. Transcriptome Analysis

[0128] To investigate the molecular-level mechanism of action of small molecule pBADS99 inhibitors (NCK and its R and S isomers), RNA sequencing (RNA-seq) was used to analyze the changes in gene expression profiles of OVCAR-3 ovarian cancer cells under different treatment conditions.

[0129] OVCAR-3 cells were treated with DMSO (negative control), R isomer, S isomer, and their racemic mixture NCK (S / R) for 18 hours, and total RNA was extracted using the Trizol method. A parallel experimental group was established where OVCAR-3 cells were transfected with pcDNA3.0 (empty vector control), pcDNA3.0-BAD WT, and pcDNA3.0-BAD S99A (dephosphorylation-mimicking mutant) plasmids for 18 hours, and cultured for an additional 12 hours after transfection to ensure stable expression. Figure 13 RNA samples were tested for concentration and integrity using Nanodrop and Agilent 2100 systems to ensure RNA quality (RIN>8.0) before subsequent sequencing. Raw Fastq sequences were quality controlled using FastQC, and Trimmomatic was used to remove low-quality reads and adapter contaminants. Clean data were aligned to the human genome reference sequence (GRCh38) using Hisat2 to generate SAM / BAM files. FeatureCounts were used to count the expression levels of each gene (expressed as FPKM), and the DESeq2 software package was used to screen for differentially expressed genes (DEGs), with thresholds set as follows: |log2 Fold Change|>1, FDR (Benjamini-Hochberg correction)<0.05. KDG screening was performed on different treatment groups (S, R, NCK) and the transfection group (S99A), and Venn diagrams were used to analyze overlapping genes. Intersection and anti-correlated gene sets were established for downregulated and upregulated KDGs, respectively.

[0130] Pearson correlation coefficients were calculated for key differentially expressed genes between each treatment group and the transfection group to assess the consistency and differences in expression patterns. Particular attention was paid to the high correlation between S99A and NCK (R = 0.810). Figure 14 ).

[0131] KEGG pathway enrichment analysis revealed that key differentially expressed genes in EOC cells transfected with the S99A vector were significantly enriched in multiple biological pathways, including oxidative phosphorylation, thermogenicity, FOXO signaling pathway, apoptosis, lysine degradation, and ubiquitin-proteasome pathway.

[0132] Oxidative phosphorylation was upregulated in EOC cells, indicating enhanced cellular energy metabolism. This pathway generates ATP via the electron transport chain in mitochondria, providing energy to the cell. Its upregulation may improve tumor cell survival, contributing to cancer cell adaptation and proliferation in hypoxic environments. Activation of the FOXO transcription factor in EOC cells may inhibit tumor cell proliferation and promote cell death by regulating the cell cycle, apoptosis, and oxidative stress responses. Activation of FOXO contributes to cancer cell senescence or apoptosis, limiting their growth and potentially increasing cellular sensitivity to chemotherapy. Activation of the apoptosis pathway was particularly prominent in EOC cells transfected with the S99A vector. Dephosphorylation of the BAD protein at the Ser99 site may enhance the apoptotic response, helping to eliminate abnormal or damaged cells, limiting cancer cell survival, and possessing potential anti-cancer effects. Changes in the lysine degradation pathway may affect tumor cell growth and survival by regulating intracellular metabolic adaptation. Activation of the ubiquitin-proteasome pathway may involve protein degradation, particularly regulating the accumulation of abnormal or damaged proteins within the cell. In summary, EOC cells transfected with the S99A vector exhibited significant changes in multiple biological pathways, particularly in energy metabolism, cell death, stress response, and metabolic adaptation. The regulation of these pathways exerts potential anti-cancer effects through mechanisms such as activating apoptosis and inhibiting proliferation. The crucial role of BAD protein Ser99 dephosphorylation in these regulatory processes provides new directions for further cancer treatment research. Figure 15 , Figure 16 ).

Claims

1. A mutant BAD protein, characterized in that, The 99th amino acid residue of the BAD protein is mutated from serine in the wild type to alanine.

2. The mutant BAD protein according to claim 1, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

1.

3. A gene encoding the mutated BAD protein of claim 1, characterized in that, The nucleotide sequence is shown in SEQ ID NO.

2.

4. A plasmid that reduces the phosphorylation level of the BAD protein at Serine 99 site, characterized in that, The gene contains the gene encoding the mutated BAD protein of claim 1, preferably the gene of claim 3.

5. The plasmid according to claim 4, characterized in that, The plasmid is obtained by inserting the gene described in claim 3 into the BamHI and EcoR1 restriction sites of the pcDNA3.0 plasmid using pcDNA3.0 as the starting plasmid.

6. Genetically engineered bacteria containing the gene of claim 3 or the recombinant plasmid of claim 4 or 5.

7. The application of a reagent that reduces the phosphorylation level of Serine 99 in the BAD protein in the preparation of antitumor drugs or drugs that improve the sensitivity of tumor cells to chemotherapy and / or radiotherapy, wherein the reagent that reduces the phosphorylation level of Serine 99 in the BAD protein is a reagent that mutates serine at position 99 of the BAD protein to alanine.

8. The application according to claim 7, characterized in that, The reagent used to reduce the phosphorylation level of BAD protein at Serine 99 is selected from any of the following: (I) The mutated BAD protein as described in claim 1; (II) The gene as described in claim 3; (III) The recombinant plasmid according to claim 4 or 5; (IV) The genetically engineered bacteria as described in claim 6.

9. A composition for treating tumors, characterized in that, The composition is selected from any one of the following: (I) The recombinant plasmid + chemotherapeutic drug as described in claim 4 or 5; (II) The genetically engineered bacteria and chemotherapy drug as described in claim 6; (III) The compound of claim 9 plus a chemotherapy drug.

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