Tβ4-17 peptide enhances the chemo-sensitivity of ovarian cancer cells to DDP by affecting NF-κB signaling pathway

Tβ4-17 peptide enhances the chemo-sensitivity of ovarian cancer cells to DDP by affecting NF-κB signaling pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Tβ4-17 peptide enhances the chemo-sensitivity of ovarian cancer cells to DDP by affecting NF-κB signaling pathway Ling Guo, Haibing Wang, Nana Li, Jing Wang, Ming Yu, Yingxu Li, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6951674/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 08 Nov, 2025 Read the published version in Medical Oncology → Version 1 posted 11 You are reading this latest preprint version Abstract Ovarian cancer is a gynecologic malignancy with high mortality and poor prognosis. Chemoresistance is a key cause of ovarian cancer recurrence and metastasis. It has been found that some bioactive peptides can inhibit the growth and metastasis of cancer cells and promote cell apoptosis, thus exerting anti-cancer effects. Tβ4-17 is a small polypeptide that we selected using ITRAQ technology, and its precursor protein is thymosin β4. This study mainly investigated its effect in combination with cisplatin (DDP) on the proliferation, migration and apoptosis of ovarian cancer resistant cells and related molecular mechanisms. Our results showed that Tβ4-17 peptide combined with DDP significantly inhibited the proliferation and migration of drug-resistant cells in ovarian cancer, promoted apoptosis, and increased the chemosensitivity of ovarian cancer cells to DDP. In addition, qRT-PCR and Western blot showed that NF-κB was significantly highly expressed in cisplatin-resistant cells of ovarian cancer. After application of NF-κB inhibitors and activators, Western blot, CCK8, EDU fluorescence proliferation assay, and cell scratch assay showed that Tβ4-17 peptide down-regulated NF-κB p65 protein expression and inhibited cell proliferation and migration. In conclusion, our study demonstrates that Tβ4-17 peptide enhances the sensitivity of ovarian cancer cells to DDP by down-regulating NF-κB expression ovarian cancer1 peptides2 Tβ4-173 DDP4 NF-κB5 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Ovarian cancer is one of the most challenging diseases in gynecological oncology. Due to the lack of specific diagnostic indicators in the early stages, most patients are diagnosed with advanced disease (Markman 2007,Wang, et al. 2021) .Platinum-based chemotherapy is an important treatment for ovarian cancer patients. Its rate of objective response is 80% in patients with ovarian cancer, but the most of them would develop platinum resistance with increasing cycles of chemotherapy, leading to recurrence, metastasis and chemotherapy failure and even death (Bristow, et al. 1999,Ledermann, et al. 2018) . Related studies have found that factors such as enhanced DNA damage repair, reduced intracellular accumulation of chemotherapeutic agents, and enhanced drug detoxification are important causes of platinum resistance (Bhattacharjee, et al. 2022,Lugones, et al. 2022) . Despite advances in drug research to reverse drug resistance, however, the toxic side effects and complexity of the mechanism of action of chemotherapeutic agents themselves have limited their clinical application. Therefore, there is an urgent need for a new agent that can improve the efficacy of chemotherapy, reduce drug resistance in ovarian cancer, and improve patient survival. Peptides are small molecules with molecular weights ranging from 500 to 5000 Da (De Souza Dutra, et al. 2023) . Endogenous peptides mostly come from the degradation of proteins and the coding of some long non-coding RNA transcripts and have functions such as immune response, cellular regulation, antibacterial and anti-inflammatory, signaling, and tumor regulation (Li, et al. 2020) . In cancer therapy, anticancer peptides have shown good application prospects and can inhibit the growth, metastasis and adhesion of cancer cells by targeting tumor blood vessels, targeting epidermal growth factor, and targeting transferrin receptor, or kill cancer cells by inducing apoptosis (Hao, et al. 2023,Ng and Lee 2020,Maijaroen, et al. 2022) . Importantly, some peptides can synergize the killing effect of chemotherapeutic drugs on cells and enhance chemosensitivity (Zhao, et al. 2016,Jaglowski, et al. 2005) . For example, the peptide Mastoparan combined with gemcitabine significantly inhibited the growth of a mouse model of breast cancer after treatment (Hilchie, et al. 2016) . Antitumor peptides offer a viable and attractive approach to the treatment of chemotherapy insensitivity or relapse. However, a few peptides have been reported in ovarian cancer. We identified an endogenous peptide, Tβ4–17 (sequence: SDKPDMAEIEKFDKSK), from the sera of cisplatin-resistant/sensitive patients with ovarian cancer, whose precursor protein is thymosin β4 (Tβ4). Tβ4 plays an important role in the organization of the cytoskeleton and functions as an actin inhibitor. Increased Tβ4 is a sensitizing factor for drug resistance in neuroblastoma and myeloma (Cheung, et al. 2015,Naeem, et al. 2023) . We hypothesized that Tβ4–17 would influence medication resistance in cancerous tumors. We therefore aimed to validate the potential role of Tβ4–17 on cisplatin resistance in ovarian cancer and to further explore the specific mechanism of its action. Materials and methods Reagents Differential peptide sequences in serum detected by ITRAQ technology (Wuhan Jinkairui Bioengineering Co., Ltd). SDKPDMAEIEKFDKSK (purity > 98%) was synthesized by Guopeptide Biotechnology Co., Ltd. DDP lyophilised powder (100mg) was purchased from Solebo Co. Rabbit polyclonal or monoclonal antibodies of human NF-κb(catalog #:T55034), β-actin (catalog #: P30002) were purchased from Abmart, China. Secondary antibody (horseradish peroxidase ‑ conjugated goat anti‑rabbit IgG, catalog #: ZB-2306) was purchased from Zhongshan jinqiao biology technology co., ltd., Beijing, China. Cell culture Human ovarian cancer cell line SKOV3 and A2780 were purchased from Nanjing Surui Medical Technology Co., Ltd. Cisplatin-resistant ovarian cancer cell lines SKOV3/DDP and A2780/DDP were purchased from Jiangsu Kaiji Biotechnology Co., Ltd. Cell lines SKOV3 and SKOV3/DDP were cultivated in McCoy's 5A (Gibco, Invitrogen, Guangzhou, China) media, whereas cell lines A2780 and A2780/DDP were cultured in RPMI-1640 (Gibco, Invitrogen, Guangzhou, China). All supplemented with heat-inactivated 10% FBS (Fetal Bovine Serum), penicillin (100 U/ml) in 5% CO2 at 37°C. The resistant strain cell line was DDP resistant, and the medium was supplied with 0.5 µg/mL DDP to maintain the DDP resistance. Cell Counting Kit-8 (CCK8) assay A2780 and A2780/DDP should be prepared at a concentration of 5 ×103 cells/100 ul, SKOV3 and SKOV3/DDP at 3.5× 103 cells/100 ul, and mix thoroughly and add 100ul to a 96-well plate. The plates were incubated in the incubator for 24 hours. The plates were grouped according to experimental needs, with 3 replicate wells per group. At the end of the experiment, cells were incubated with 10 µL of CCK-8 reagent (sigma Aldrich; Merck KGaA) for 2h at 37°C. The results were detected at 450 nm on a microplate reader (TECAN infinite; TECAN Austria GmbH, groedig, Austria). The percent viability of cells was calculated using the following formula. Cell viability (%) = [A (drug added) -A (blank)]/ [A (0 drug added) -A (blank)] × 100%. Each experiment was triplicated independently. Plane clone formation assay The control group, 5umol/LDDP group and 5umol/LDDP + 5umol/L Tβ4-17peptide group after 48 hours of intervention were selected for colony formation experiments based on the results of the preliminary CCK8 cell viability assay. The above intervention groups were counted separately and the cells (500 cells/well) were seeded into 6-well plates. After cultured for 14 days, colonies were fixed with 4% paraformaldehyde at room temperature for 20 min and stained with crystal violet for 20 min, and the numbers of visible colonies were counted. EDU fluorescence proliferation assay Cell configuration concentration was the same as CCK8 assay. The groups were grouped according to the experimental needs and incubated for 2 hours after adding 50ul of EDU working solution. Remove the EDU working solution, add 100ul of 4% paraformaldehyde for fixation for 15-30min, and then incubate with cell permeabilization solution for 10-15min in room temperature environment. The Click reaction solution was prepared according to the instructions and the number of samples to be tested, 50ul was taken and added to the wells to be tested and then incubated for 1 hour. Each well received 100 L of the prepared 1X Hoechst solution and was incubated at room temperature for 20 minutes and photographed under a fluorescence microscope. Wound healing assay Cells were cultured to confluence in 6-well plates. A 200 µL pipette tip was used to scratch in each well. Cells were rinsed 3 times with PBS, and then the corresponding culture solution containing the drug to be tested was added separately according to the experimental grouping requirements and placed in the incubator. The 6-well plates were taken out at 0 and 48 hours of drug addition to take images, which were then analyzed by Image J. Transwell assays Cells treated in advance with 5µmol/L DDP, 5µmol/L DDP + 5µmol/LTβ4–17 peptide for 48 hours were resuspended in serum-free cell culture medium and counted as 8×104 cells, which were categorized into control, DDP, and DDP + Tβ4-17peptide groups. In a 24-well plate, 800ul of culture medium containing 20% FBS was first added, followed by 200ul of serum-free cell suspension evenly dripped into the chamber, and incubated in the incubator. After 24 hours, the culture medium was discarded, fixed for 15–30 min, and then stained with crystal violet for 20 min. The cells in the lower chamber were observed under the microscope, photographed and counted. TUNEL assays Cell configuration concentration was the same as CCK8 assay. Treated with 5µmol/L DDP, 5µmol/L DDP + 5µmol/L Tβ4–17 peptide. After 48 h, 4% paraformaldehyde for fixation for 20 min was added and incubated for 5 min at room temperature with potent permeabilization solution. TUNEL assay solution was prepared according to the instructions and the number of experimental samples. 50µl TUNEL assay solution was added to the wells of the samples to be tested and incubated for 1 hour. Then add 100ul DAPI staining solution, incubate for 10min at room temperature. PBS was washed 3 times and photographed under fluorescence microscope observation. Flow Cytometry Analysis Cells were treated with different drugs (0, 5µmol/L DDP, 5µmol/L DDP + 5µmol/LTβ4–17) for 48 h, and trypsin digestion was added. The precipitate was gently resuspended with pre-cooled PBS and counted, and 1×105 cells/ml resuspended were taken, centrifuged at 1000 rpm for 5 min, and the cells were resuspended by adding Annexin V-FITC conjugate and gently mixed. Add 10µl of propidium iodide staining solution, incubate for 20min at room temperature, and then test on the machine. Reverse transcription-quantitative PCR (RT-qPCR) The primers were designed by Primer 6.0 software according to genes sequences searched by Primer-Bank, and synthetized by Shanghai Shenggong Biological Co., Ltd.. Ovarian cancer cells were harvested using RNAkey ™ Reagent, RNA was reverse-transcribed to cDNA using EvoM-MLV Reverse Transcription Kit (Accurate Biotechnology) following the manufacturer’s instructions. The reverse transcription reaction conditions were 37°C for 15min and 85°C for 5sec. Real time PCR reaction conditions were as follows: 95°C pre-denaturation 30 s; 95°C ×5 s, 60°C×30 s (40 cycles). GAPDH was used as the housekeeping gene. For target gene, mRNA expression levels were calculated using the 2‑ΔΔCt method (ΔCt = target gene Ct ‑ GAPDH Ct value). The primer sequence is as follows: GAPDH, 5’-GAGTCAACGGATTTGGTCGT-3' and 5’-TTGATTTTGGAGGGATCTCG-3'. NF-κB, 5’-AATCCAGTGTGTGAAGAAGC-3' and 5’- GCTGCTCTTCTATAGGAACT-3'. Western Blot assay Cells were collected and lysed after 48h of treatment with different drugs. Equal amounts of protein lysate were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to PVDF membranes. 5% skimmed milk was closed for 2 hours and incubated with primary antibodies overnight at 4°C. The primary antibody used in this study are as follows: Anti-β-actin Rabbit Antibody (Abmart, P30002), Anti-NF-κB (P65) Rabbit Antibody (Abmart, T55034). Then, horseradish peroxidase-conjugated secondary antibody (HRP-conjugated goat anti-rabbit IgG). After incubation at 37 ° C for 2 h, protein bands were visualized by enhanced chemiluminescence Plus kit (Beyotime, Shanghai, China). Images were acquired by an automated chemiluminescence image analysis system (Tanon, Shanghai, China). Statistical analysis Image J software was used for cell migration and protein gray value analysis, and GraphPad Prism9.0 performed statistical analysis and mapping of the data. The experimental results of each group were repeated three times. Statistical results All values were expressed as mean ± standard deviation (Means ± SD), and the comparison of samples between two groups was performed by t-test. P < 0.05 was considered as a significant difference. Results Screening and expression validation of Tβ4–17 peptide in cisplatin-resistant/sensitive sera of ovarian cancer Our group successfully screened 41 differentially expressed peptide sequences using ITRAQ technology in the early stage (Li 2025) .Analysis of the basic characteristics of the differential peptides revealed that the isoelectric points of these peptides were in the range of 3.0–12.5, but mainly in the acidic range (Fig. 1 A). The molecular weights of these peptides were mainly in the range of 800–1600 Da, which was consistent with the principle of peptide identification (Fig. 1 B). In order to further understand the physicochemical properties and related information of Tβ4–17 peptide, we used the ExPASy and ProtParam online programs and discovered: Tβ4–17 consists of 16 amino acids, with molecular weight of 1,868.09 Da, isoelectric point of 4.86, and instability index of 44.26, which is an unstable polypeptide, the fat-solubility index is 30.63 and the total average hydrophilicity index was − 1.581 (Table 1 ). This suggests that Tβ4–17 is a hydrophilic small molecule peptide. We applied the PRM technique to further define the expression of Tβ4–17 polypeptide in cisplatin-resistant/sensitive sera of ovarian cancer (Table 2 , Fig. 1 C). The results showed that Tβ4–17 peptide was significantly under-expressed in cisplatin-resistant serum of ovarian cancer. Table 1 Physicochemical Properties and Related Information of the Tβ4–17 Peptide. Physicochemical Characteristics Value Number of amino acids 1868.09 Theoretical PI 4.86 Instability index 44.26 Aliphatic index 30.63 Grand average of hydropathicity -1.581 Table 2 Relative expression of target peptides in cisplatin-resistant/sensitive serum of ovarian cancer. Sample R (Drug-resistant serum values) S (Sensitive serum values) 1 952434 1287873 2 844393 1875993 3 804709 1641410 4 969653 1940420 5 1255356 1693780 6 983715 973706 7 1171817 2095880 8 921588 1179725 9 1573706 968543 10 1588882 1724969 11 874026 1684198 12 1418046 1392530 Identification of Cisplatin-resistant Ovarian Cancer Cells A2780/DDP and SKOV3/DDP We applied the CCK8 assay to detect whether A2780/DDP and SKOV3/DDP are resistant to drugs. The results showed that the number of drug-resistant and parental cells surviving decreased with the increase of DDP concentration. Moreover, the survival rate of parental cells was lower than that of drug-resistant cells at the same concentration of DDP (Fig. 1 D, G). According to the absorbance values measured by CCK8, the resistance index were 5.28 and 2.08 for A2780/DDP and 5.06 and 2.51 for SKOV3/DDP at 24 and 48 hours(Table 3 ). The above results confirmed that both cells were resistant. Table 3 IC50 values and RI values of ovarian cancer parental cells and drug-resistant cells at 24H and 48H. Time A2780 A2780/DDP SKOV3 SKOV3/DDP 24H-IC50(µmol/L) 48H-IC50(µmol/L) 24H-RI 48H-RI 5.269 4.553 27.82 9.512 5.28 2.08 6.95 5.64 35.17 14.17 5.06 2.51 Tβ4–17 enhanced DDP-inhibited of human ovarian cancer cell viability and migration Our experiments have confirmed that 5µmol/L of DDP is an optimal concentration for the experiment. We tested the effects of different concentrations of Tβ4–17 (1µmol/L, 5µmol/L, 25µmol/L) combined with DDP on cell proliferation. CCK8 results showed that Tβ4–17 combined with DDP resulted in a significant dose-dependent decrease in ovarian cancer parental and resistant cell viability compared to DDP alone. And the inhibition of viability in the drug-resistant cells was stronger than that of parental cells (Fig. 2 A-D). Based on this result, we set the concentration of Tβ4–17 to 5µmol/L for subsequent cell experiments. Then, we carried out plate clone formation (Fig. 2 E) and EDU tests (Fig. 2 F, G) to confirm the growth inhibition impact of T4-17 on ovarian cancer cells. The results were consistent with the CCK8 assay, with the lowest number of viable cells in the 5µmol/L Tβ4–17 combined with DDP treatment group. These results indicate that Tβ4–17 reduces the resistance of ovarian cancer cells to DDP. To explore whether Tβ4–17 combined with DDP caused migration inhibition. We first examined cell migration rates using a cell scratch assay, which showed that Tβ4–17 combined with DDP treatment slowed cell convergence to closure (Fig. 2 H, I). This migration inhibition was stronger for ovarian cancer resistant cells. Subsequently, we used transwell assay to detect cell migration after co-treatment. When Tβ4–17 was coupled with DDP therapy, the number of cells passing through the microtiter wells was decreased after 48 hours compared to the DDP treatment group alone (Fig. 2 J). This suggests that Tβ4–17 enhances the migration inhibition of ovarian cancer cells by DDP. Tβ4–17 increased DDP-induced apoptosis in human ovarian cancer cells We used the TUNEL technique to detect apoptosis in drug-resistant cells in ovarian cancer to investigate the effect of Tβ4–17 coupled with DDP on apoptosis. The results showed that the number of apoptotic cells was significantly increased in the Tβ4–17 combined with DDP group compared with the DDP alone group (Fig. 3 A, B). Flow cytometry to further analyze revealed that the apoptosis rates of DDP alone treated A2780/DDP and SKOV3/DDP cells were 25.3% and 24.5%, respectively, whereas the apoptosis rates after the addition of Tβ4–17 were 56.2% and 51.8%, respectively (Fig. 3 C, D). The result suggests that Tβ4–17 enhances apoptosis induction by DDP. Tβ4–17 regulated NF-κB expression To investigate whether NF-κB expression levels are associated with drug resistance, we examined NF-κB expression in ovarian cancer parental cells and drug-resistant cells by qRT-PCR. The results showed that the expression level of NF-κB mRNA in drug-resistant cells was significantly higher than that in parental cells (Fig. 4 A). Western blot results showed that NF-κB p65 protein was significantly overexpressed in ovarian cancer resistant cells compared to ovarian cancer parental cells (Fig. 4 B). Our result indicates that high expression of NF-κB p65 protein is associated with drug resistance in ovarian cancer. Given the involvement of NF-κB in ovarian cancer drug resistance, we examined the expression level of NF-κB p65 protein in ovarian cancer cells after Tβ4–17 combined with DDP. Western blot results showed that the NF-κB p65 protein content was decreased after Tβ4–17 combined with DDP acted on the cells compared to DDP treatment alone (Fig. 4 C, D). It suggests that Tβ4–17 combined with DDP can downregulate the activation of the NF-κB pathway, which partially reversed cellular resistance to drug. Tβ4–17 enhances chemosensitivity of ovarian cancer cells to DDP through the NF-κB pathway To further investigate whether Tβ4–17 regulates ovarian cancer cells through the NF-κB signaling pathway, PDTC was selected as an NF-κB inhibitor. Western blot analysis revealed that PDTC significantly inhibited the expression of the NF-B p65 protein in drug-resistant ovarian cancer cells. Compared with the decrease in NF-κB p65 protein expression induced by the DDP + PDTC group, interestingly, it furthermore decrease after the addition of Tβ4–17, suggesting that Tβ4–17 can effectively down-regulate NF-κB p65 protein expression (Fig. 5 A). We also tested the effect on cell proliferation after application of PDTC. The results of CCK8 assay (Fig. 5 B) and EDU fluorescence assay (Fig. 5 C, D) showed that the PDTC group alone inhibited cell proliferation, suggesting that inhibition of NF-κB expression in the cells slowed down cell growth. The highest cell growth inhibition rate was achieved with the addition of Tβ4–17 compared to the DDP combined with PDTC group. In addition, we examined the migratory capacity of the cells. The results showed that the cell migration inhibition effect was most obvious in the group treated with the addition of Tβ4–17 (Fig. 5 E, F). Taken together, these results suggest that Tβ4–17 inhibits the proliferation and migration of drug-resistant cells of ovarian cancer by down-regulating the expression of NF-κB. To further confirm the relationship between Tβ4–17 and the NF-κB pathway, TNF-α was used as an activator of NF-κB. Western blot results showed that TNF-α could effectively activate the expression of NF-κB p65 protein in drug-resistant cells of ovarian cancer. NF-κB p65 protein was significantly decreased after application of Tβ4–17 compared to the DDP + TNF-α group (Fig. 6 A). It is suggested that Tβ4–17 peptide can effectively reverse the expression of NF-κB p65 protein after application of TNF-α. We also analyzed the effect on cell proliferation after application of TNF-α. The results of CCK8 assay (Fig. 6 B) and EDU fluorescence assay (Fig. 6 C, D) showed that TNF-α alone had no significant effect on cell proliferation. The proliferative capacity of the cells in the DDP + TNF-α group was significantly decreased, but the most significant decrease was observed with the addition of Tβ4–17 peptide. In addition, the results of cell scratch assay showed that TNF-α had no significant effect on cell migration. The ability of cells in the DDP + TNF-α group to aggregate toward the middle was slowed, but the number of resistant cells moving toward the middle was significantly reduced by the addition of Tβ4–17 peptide (Fig. 6 E, F). Taken together, Tβ4–17 peptide can inhibit cell proliferation and migration through down-regulation of the NF-κB pathway, which in turn enhances the chemo-sensitivity of cells to DDP. Discussion Ovarian cancer remains one of the deadliest malignancies among gynecologic cancers due to the lack of early symptoms and the difficulty of clinical diagnosis (Ayhan, et al. 2023) . DDP-based platinum-based drugs are commonly used chemotherapeutic agents for the treatment of ovarian cancer. However, long-term and high-dose administration of DDP can lead to serious side effects (Zangouei and Moghbeli 2021) . Importantly, the development of drug resistance is frequently observed in patients with ovarian cancer, making DDP less effective and leading to failure of tumor suppression and recurrence (Dall'Acqua, et al. 2017) . Peptides provide us with valuable information, and their expression can be detected throughout the body in organs and tissues as well as in cells and body fluids (Ghose, et al. 2022,Tammen, et al. 2007) . Several studies have confirmed that peptides can be used to diagnose or treat various diseases (Guo, et al. 2023) . Pan et al (Pan, et al. 2022) found that the endogenous peptide PDHPS1 disrupts YAP signaling by interacting with PTPA in ovarian cancer, thereby inhibiting the growth of ovarian cancer cells in vitro and in vivo. In addition, peptides also play an active role in chemosensitivity. GTGKT peptide reduces the angiogenic, tumorigenic and metastatic potential of anticancer-resistant cancer cells and enhances the apoptotic effect and sensitivity of anticancer drugs by inhibiting the binding of tumor-associated genes to the cyclin D1 promoter sequence (Kim, et al. 2017) . In view of the important role played by peptides in tumor therapy, our group used ITRAQ technology to screen for differentially expressed bioactive peptides from cisplatin-resistant/sensitive sera of ovarian cancer (Li 2025) . Among them, the relatively low expression of Tβ4–17 in drug-resistant sera of ovarian cancer and its possible association with drug resistance. The findings of this study indicate that Tβ4–17 enhances the cytotoxicity of DDP against ovarian cancer cells, inhibits migration, promotes apoptosis, and increases sensitivity of ovarian cancer cells to cisplatin. Due to the significantly higher cytotoxic effects of Tβ4–17 and DDP on drug-resistant ovarian cancer cells compared to sensitive cells, it indicates that Tβ4–17 can synergistically enhance the effects of DDP. Our results also suggest that Tβ4–17 may enhance the sensitivity of ovarian cancer cells to DDP through the NF-κB signaling pathway, but the related mechanisms require further investigation. The drug resistance mechanism of ovarian cancer is highly complex, involving multiple genes and pathways (Freimund, et al. 2018) . In recent years, the role of the NF-κB signaling pathway in drug resistance in ovarian cancer has drawn extensive attention. In most cell types, NF-κB exists in an inactive state in the cytoplasm and forms a trimeric complex with the inhibitory factor IκB, thus transcriptionally inactive before the cell is activated by relevant stimuli (Guan, et al. 2022) . Activation of the NF-κB pathway has been reported in various types of tumors, leading to enhanced expression of anti-apoptotic proteins, increased secretion of pro-inflammatory cytokines, and promotion of MDR (Yuan, et al. 2020,Tan, et al. 2019) . Most chemotherapeutic agents induce some form of DNA damage, causing activation of the NF-κB signalling pathway. Yan et al (Yan, et al. 2014) found that activation of the ATM/NF-κB pathway increased chemoresistance in lung cancer cells and enhanced the expression of Bcl-2, Mcl-1, Bcl-xl and ABCG2. Hu et al (Hu, et al. 2021) demonstrated that NF-κB activation was associated with glioma drug resistance, leading to temozolomide resistance. Conversely, interfering with NF-κB activity enhances the susceptibility of transplanted tumours to drug resistance, resulting in a reduction in tumour size and weight (Liu, et al. 2021) . Overexpression of cytosolic NF-κB in ovarian cancer has been reported to be associated with advanced FIGO staging, residual lesions ≥ 1 cm, low histological grade, chemotherapy resistance as well as platinum resistance and refractory disease, as well as a more aggressive ovarian cancer phenotype (Kan, et al. 2020) cells. In this study, we observed increased transcriptional activity of NF-kB in drug-resistant ovarian cancer cells compared to parental cells. This result is consistent with previous findings in ovarian cancer cells (Yan, et al. 2017) . PDTC acts as an inhibitor of NF-κB and effectively inhibits its expression. Similarly, in our experiments, PDTC reduced the expression of NF-κB in drug-resistant cells of ovarian cancer, and cell viability was significantly inhibited. Importantly, the addition of Tβ4–17 co-treatment significantly enhanced the inhibitory effect of cisplatin. In contrast, TNF-α as an NF-κB pathway activator activated NF-κB expression in cells but had no significant effect on cell viability. Previous studies have reported that TNF-α co-administered with PTX enhances the latter's apoptotic response, and its cytotoxicity can be increased in combination with DDP (Noonan, et al. 2020) . We demonstrated that TNF-α enhanced the cytotoxicity of DDP, but this cytotoxic effect reached its maximum with the addition of Tβ4–17. Taken together, we demonstrated that Tβ4–17 enhanced the sensitivity of human ovarian cancer cell lines (A2780, SKOV3) to their DDP-resistant cell lines (A2780/DDP, SKOV3/DDP). In addition, NF-κB was shown to be associated with resistance to cisplatin chemotherapy in ovarian cancer. In this study, we verified the alteration of NF-κB protein expression by Tβ4–17 by applying NF-κB inhibitor (PDTC) and NF-κB activator (TNF-α), respectively. It is speculated that Tβ4–17 may increase the sensitivity of ovarian cancer cells to DDP by down-regulating the NF-κB signalling pathway. These data help to understand the role of Tβ4–17 in chemotherapy and may contribute to the development of an effective therapeutic strategy to overcome DDP resistance in ovarian cancer. Declarations Consent for publication : Not applicable Availability of data and materials: Not applicable Competing interests : The authors declare that they have no competing interests. Funding ： The study was supported by the Harbin Medical University Cancer Hospital Haiyan Research Foundation [grant number JJZD2022-01] Authors' Contributions ： Ling Guo wrote the main manuscript text and performed experiments with the assistance of Jing Wang and Nana Li; Yu Ming collected serum samples; Yingxu Li and Peihua Yan performed the statistical analysis; Yajuan Su and Lichen Teng corrected and reviewed the article before submission. References Markman, M. 2007. 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"Targeting CD133 reverses drug-resistance via the AKT/NF-κB/MDR1 pathway in colorectal cancer." BRIT J CANCER., 122 (9), 1342-1353. Tan, C., Liu, L., Liu, X., Qi, L., Wang, W., Zhao, G. et al. 2019. "Activation of PTGS2/NF-κB signaling pathway enhances radiation resistance of glioma." Cancer medicine, 8 (3), 1175-1185. Yan, H., Huang, X., Ke, S., Jiang, Y., Zhang, Y., Wang, Y. et al. 2014. "Interleukin 6 augments lung cancer chemotherapeutic resistance via ataxia-telangiectasia mutated/NF-kappaB pathway activation." Cancer science, 105 (9), 1220-1227. Hu, Y.H., Jiao, B.H., Wang, C.Y. and Wu, J.L. 2021. "Regulation of temozolomide resistance in glioma cells via the RIP2/NF-kappaB/MGMT pathway." CNS Neurosci Ther, 27 (5), 552-563. Liu, Y., Jia, Y., Liu, S. and Ma, J. 2021. "FSTL1 increases cisplatin sensitivity in epithelial ovarian cancer cells by inhibition of NF-κB pathway." CANCER CHEMOTH PHARM. , 87 (3), 405-414. Kan, Y., Liu, J. and Li, F. 2020. "High Expression of Nuclear Transcription Factor-κB is Associated with Cisplatin Resistance and Prognosis for Ovarian Cancer." Cancer Manag Res. , 12, 8241-8252. Yan, X., Zhang, Y., Zhang, J., Zhang, L., Liu, Y., Wu, Y. et al. 2017. "p62/SQSTM1 as an oncotarget mediates cisplatin resistance through activating RIP1-NF-κB pathway in human ovarian cancer cells." Cancer science.108 (7), 1405-1413. Noonan, A., Cousins, A., Anderson, D., Zeligs, K., Bunch, K., Hernandez, L. et al. 2020. "Matrix Drug Screen Identifies Synergistic Drug Combinations to Augment SMAC Mimetic Activity in Ovarian Cancer." Cancers.12 (12). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 08 Nov, 2025 Read the published version in Medical Oncology → Version 1 posted Editorial decision: Revision requested 23 Sep, 2025 Reviews received at journal 20 Sep, 2025 Reviews received at journal 12 Sep, 2025 Reviewers agreed at journal 06 Sep, 2025 Reviewers agreed at journal 02 Sep, 2025 Reviews received at journal 05 Aug, 2025 Reviewers agreed at journal 21 Jul, 2025 Reviewers invited by journal 20 Jul, 2025 Editor assigned by journal 22 Jun, 2025 Submission checks completed at journal 22 Jun, 2025 First submitted to journal 22 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6951674","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":488747655,"identity":"b8890bf2-3a34-4768-a765-b56a9911e633","order_by":0,"name":"Ling Guo","email":"","orcid":"","institution":"Harbin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ling","middleName":"","lastName":"Guo","suffix":""},{"id":488747656,"identity":"cc3b073b-1621-4454-bbaa-005138666070","order_by":1,"name":"Haibing Wang","email":"","orcid":"","institution":"Harbin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Haibing","middleName":"","lastName":"Wang","suffix":""},{"id":488747657,"identity":"94c79a87-8fed-4929-ae60-1e0d9ed31c4d","order_by":2,"name":"Nana Li","email":"","orcid":"","institution":"Harbin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Nana","middleName":"","lastName":"Li","suffix":""},{"id":488747658,"identity":"226fefa1-1907-4315-9779-25a3c5ab4ec2","order_by":3,"name":"Jing Wang","email":"","orcid":"","institution":"Harbin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Wang","suffix":""},{"id":488747659,"identity":"9c78bb77-625b-4a25-b639-45b97498de61","order_by":4,"name":"Ming Yu","email":"","orcid":"","institution":"Harbin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ming","middleName":"","lastName":"Yu","suffix":""},{"id":488747660,"identity":"05a46681-f1d4-44fa-ae42-7bfa4573747b","order_by":5,"name":"Yingxu Li","email":"","orcid":"","institution":"Harbin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yingxu","middleName":"","lastName":"Li","suffix":""},{"id":488747661,"identity":"ed7f2c8f-f6da-44c5-84e4-c1989d3cf56a","order_by":6,"name":"Peihua Yan","email":"","orcid":"","institution":"Harbin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Peihua","middleName":"","lastName":"Yan","suffix":""},{"id":488747662,"identity":"4e43d900-3119-461d-8863-83f218d3f34d","order_by":7,"name":"Yajuan Su","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxklEQVRIiWNgGAWjYBACfv7GhgMfGxgSwDweYrRIzjjceHAmSVoMDqQ3H+YlSQvDgYMNh2133MvTnZHA+OBtG4O8OSEdjM2NDYdzzxQXm91IYDac28ZguLOBgBZmBqAtuW0JidtuJLBJ87YxJBgcIKCFjSGx4bAlRAv7b6K08IC0MEJtYSZKi4TEwYaDvW0JxWZnHjZLzjknYbiBkBb78+2PP/xsS8gzO5588MObMht5grYgAcYGkK3Eqx8Fo2AUjIJRgBsAAJJWSTpjLIDnAAAAAElFTkSuQmCC","orcid":"","institution":"Harbin Medical University","correspondingAuthor":true,"prefix":"","firstName":"Yajuan","middleName":"","lastName":"Su","suffix":""},{"id":488747663,"identity":"e9c344e8-18b9-46e4-8e1c-689b9dad6b94","order_by":8,"name":"Lichen Teng","email":"","orcid":"","institution":"Harbin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Lichen","middleName":"","lastName":"Teng","suffix":""}],"badges":[],"createdAt":"2025-06-23 00:38:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6951674/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6951674/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12032-025-03106-4","type":"published","date":"2025-11-08T15:57:04+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":87420719,"identity":"f5ade635-a800-4a6b-b636-7a1196543701","added_by":"auto","created_at":"2025-07-23 15:30:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":407952,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScreening and identification of Tβ4-17 and detection of drug-resistant cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Isoelectric Point Distribution of Peptides. (B) Molecular weight distribution of peptides. (C) Tβ4-17 expression in cisplatin-resistant/sensitive serum of ovarian cancer detected by PRM technique. (D, E) CCK8 detects A2780/DDP resistance at 24H and 48H. (F, G) CCK8 detects SKOV3/DDP resistance at 24H and 48H. The data were presented as the mean ± SD (n = 3), *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003cem\u003et\u003c/em\u003e test.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6951674/v1/fdd507721d1809f48c932633.png"},{"id":87421407,"identity":"991efa65-fd65-49b6-b52b-b3d563654db1","added_by":"auto","created_at":"2025-07-23 15:38:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4899650,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTβ4-17 enhanced DDP inhibition of human ovarian cancer cell viability and migration.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-D) The effects of different concentrations of Tβ4-17 peptide bound to DDP on the proliferation of A2780, SKOV3, A2780/DDP and SKOV3/DDP were examined by CCK8. (E) Colony formation profiles of A2780, A2780/DDP, SKOV3 and SKOV3/DDP cells treated with Tβ4-17 peptide combined with DDP. (F, G) Fluorescence proliferation plot of A2780, A2780/DDP, SKOV3 and SKOV3/DDP cells treated with Tβ4-17 peptide combined with DDP. The data were presented as the mean ± SD (n = 3), *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 compared with cell lines intervened by DDP alone in the same time. (H, I) Migration profile of A2780, A2780/DDP, SKOV3 and SKOV3/DDP cells treated with Tβ4-17 peptide combined with DDP. (J) Transwell migration profiles and migration rates of A2780, A2780/DDP, SKOV3 and SKOV3/DDP cells under different treatment conditions. The data were presented as the mean ± SD (n = 3), *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, \u003cem\u003et\u003c/em\u003e test.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6951674/v1/250f39f16ef0d88fe4586b3e.png"},{"id":87421404,"identity":"222956ef-e548-42d3-bba5-d9e43cb06667","added_by":"auto","created_at":"2025-07-23 15:38:32","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":979907,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTβ4-17 increased DDP-induced apoptosis inhuman ovarian cancer cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A, B) TUNEL assay was used to detect the apoptotic map and histogram of apoptotic rate of A2780/DDP, SKOV3/DDP cells treated with Tβ4-17 combined with DDP. (C) Representative apoptosis results of A2780/DDP, SKOV3/DDP cells treated with Tβ4-17 in combination with DDP analyzed by flow cytometry. (D) Histogram of apoptosis detected by flow cytometry. The data were presented as the mean ± SD (n = 3), *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003cem\u003et\u003c/em\u003e test.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6951674/v1/159e55a7d4c614052e5652ac.png"},{"id":87420723,"identity":"d47346af-e1ed-4826-8672-2ebe670ca1be","added_by":"auto","created_at":"2025-07-23 15:30:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":606817,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTβ4-17 regulated NF-κB expression.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) NF-κB mRNA expression was analyzed using qRT-PCR. (B) Protein map and relative expression of NF-κB protein. (C, D) Image of NF-κB protein expression and relative expression in A2780, A2780/DDP, SKOV3 and SKOV3/DDP cells treated with Tβ4-17 in combination with DDP. The data were presented as the mean ± SD (n = 3), *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, \u003cem\u003et\u003c/em\u003etest.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6951674/v1/e91f62cb2019767a8aa8d230.png"},{"id":87420725,"identity":"ca6c4487-855b-42f6-921e-8f0387ebe570","added_by":"auto","created_at":"2025-07-23 15:30:32","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2285025,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of Tβ4-17 on drug-resistant cells of ovarian cancer after application of PDTC.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) NF-κB protein expression map and relative expression of NF-κB protein in Tβ4-17-treated A2780/DDP and SKOV3/DDP cells after application of NF-κB inhibitors. (B) CCK8 assay was used to detect the proliferation of A2780/DDP and SKOV3/DDP cells under different treatment conditions. (C, D) Fluorescence proliferation maps and relative expression of Tβ4-17-treated A2780/DDP, SKOV3/DDP cells after application of NF-κB inhibitors. (E, F) Scratch assay was performed to detect the migration profiles and relative expression of Tβ4-17-treated A2780/DDP, SKOV3/DDP cells after application of NF-κB inhibitors. The data were presented as the mean ± SD (n = 3), *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, \u003cem\u003et\u003c/em\u003e test.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6951674/v1/b85fb302b0d3670d1a290cbf.png"},{"id":87420720,"identity":"ed6c6068-c23a-4a4e-80bc-f77471aad6fe","added_by":"auto","created_at":"2025-07-23 15:30:32","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2276084,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of Tβ4-17 on drug-resistant cells of ovarian cancer after application of TNF-α.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Images of NF-κB protein expression and relative expression in Tβ4-17-treated A2780/DDP, SKOV3/DDP cells following NF-κB agonist application. (B) CCK8 assay applied to the proliferation of Tβ4-17-treated A2780/DDP and SKOV3/DDP cells after NF-κB agonists. (C, D) Proliferation fluorogram and relative expression of A2780/DDP, SKOV3/DDP cells under different treatment conditions. (E, F) Scratch assay was performed to detect the migration profiles and relative expression of Tβ4-17-treated A2780/DDP, SKOV3/DDP cells after application of NF-κB agonist. The data were presented as the mean ± SD (n = 3), ns: no significance, *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001, \u003cem\u003et\u003c/em\u003e test.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6951674/v1/716271e684dc183116ae0e55.png"},{"id":95563940,"identity":"4c2cfa6b-352c-4a77-9430-0518da5de5a9","added_by":"auto","created_at":"2025-11-10 16:04:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12142131,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6951674/v1/292fc0b2-ba8a-4326-a7e7-fd8725d7b519.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Tβ4-17 peptide enhances the chemo-sensitivity of ovarian cancer cells to DDP by affecting NF-κB signaling pathway","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOvarian cancer is one of the most challenging diseases in gynecological oncology. Due to the lack of specific diagnostic indicators in the early stages, most patients are diagnosed with advanced disease\u003csup\u003e(Markman 2007,Wang, et al. 2021)\u003c/sup\u003e.Platinum-based chemotherapy is an important treatment for ovarian cancer patients. Its rate of objective response is 80% in patients with ovarian cancer, but the most of them would develop platinum resistance with increasing cycles of chemotherapy, leading to recurrence, metastasis and chemotherapy failure and even death\u003csup\u003e(Bristow, et al. 1999,Ledermann, et al. 2018)\u003c/sup\u003e. Related studies have found that factors such as enhanced DNA damage repair, reduced intracellular accumulation of chemotherapeutic agents, and enhanced drug detoxification are important causes of platinum resistance\u003csup\u003e(Bhattacharjee, et al. 2022,Lugones, et al. 2022)\u003c/sup\u003e. Despite advances in drug research to reverse drug resistance, however, the toxic side effects and complexity of the mechanism of action of chemotherapeutic agents themselves have limited their clinical application. Therefore, there is an urgent need for a new agent that can improve the efficacy of chemotherapy, reduce drug resistance in ovarian cancer, and improve patient survival.\u003c/p\u003e\u003cp\u003ePeptides are small molecules with molecular weights ranging from 500 to 5000 Da\u003csup\u003e(De Souza Dutra, et al. 2023)\u003c/sup\u003e. Endogenous peptides mostly come from the degradation of proteins and the coding of some long non-coding RNA transcripts and have functions such as immune response, cellular regulation, antibacterial and anti-inflammatory, signaling, and tumor regulation\u003csup\u003e(Li, et al. 2020)\u003c/sup\u003e. In cancer therapy, anticancer peptides have shown good application prospects and can inhibit the growth, metastasis and adhesion of cancer cells by targeting tumor blood vessels, targeting epidermal growth factor, and targeting transferrin receptor, or kill cancer cells by inducing apoptosis\u003csup\u003e(Hao, et al. 2023,Ng and Lee 2020,Maijaroen, et al. 2022)\u003c/sup\u003e. Importantly, some peptides can synergize the killing effect of chemotherapeutic drugs on cells and enhance chemosensitivity\u003csup\u003e(Zhao, et al. 2016,Jaglowski, et al. 2005)\u003c/sup\u003e. For example, the peptide Mastoparan combined with gemcitabine significantly inhibited the growth of a mouse model of breast cancer after treatment\u003csup\u003e(Hilchie, et al. 2016)\u003c/sup\u003e. Antitumor peptides offer a viable and attractive approach to the treatment of chemotherapy insensitivity or relapse. However, a few peptides have been reported in ovarian cancer.\u003c/p\u003e\u003cp\u003eWe identified an endogenous peptide, Tβ4\u0026ndash;17 (sequence: SDKPDMAEIEKFDKSK), from the sera of cisplatin-resistant/sensitive patients with ovarian cancer, whose precursor protein is thymosin β4 (Tβ4). Tβ4 plays an important role in the organization of the cytoskeleton and functions as an actin inhibitor. Increased Tβ4 is a sensitizing factor for drug resistance in neuroblastoma and myeloma\u003csup\u003e(Cheung, et al. 2015,Naeem, et al. 2023)\u003c/sup\u003e. We hypothesized that Tβ4\u0026ndash;17 would influence medication resistance in cancerous tumors. We therefore aimed to validate the potential role of Tβ4\u0026ndash;17 on cisplatin resistance in ovarian cancer and to further explore the specific mechanism of its action.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eReagents\u003c/h2\u003e\u003cp\u003eDifferential peptide sequences in serum detected by ITRAQ technology (Wuhan Jinkairui Bioengineering Co., Ltd). SDKPDMAEIEKFDKSK (purity\u0026thinsp;\u0026gt;\u0026thinsp;98%) was synthesized by Guopeptide Biotechnology Co., Ltd. DDP lyophilised powder (100mg) was purchased from Solebo Co. Rabbit polyclonal or monoclonal antibodies of human NF-κb(catalog #:T55034), β-actin (catalog #: P30002) were purchased from Abmart, China. Secondary antibody (horseradish peroxidase ‑ conjugated goat anti‑rabbit IgG, catalog #: ZB-2306) was purchased from Zhongshan jinqiao biology technology co., ltd., Beijing, China.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCell culture\u003c/h3\u003e\n\u003cp\u003eHuman ovarian cancer cell line SKOV3 and A2780 were purchased from Nanjing Surui Medical Technology Co., Ltd. Cisplatin-resistant ovarian cancer cell lines SKOV3/DDP and A2780/DDP were purchased from Jiangsu Kaiji Biotechnology Co., Ltd. Cell lines SKOV3 and SKOV3/DDP were cultivated in McCoy's 5A (Gibco, Invitrogen, Guangzhou, China) media, whereas cell lines A2780 and A2780/DDP were cultured in RPMI-1640 (Gibco, Invitrogen, Guangzhou, China). All supplemented with heat-inactivated 10% FBS (Fetal Bovine Serum), penicillin (100 U/ml) in 5% CO2 at 37\u0026deg;C. The resistant strain cell line was DDP resistant, and the medium was supplied with 0.5 \u0026micro;g/mL DDP to maintain the DDP resistance.\u003c/p\u003e\n\u003ch3\u003eCell Counting Kit-8 (CCK8) assay\u003c/h3\u003e\n\u003cp\u003eA2780 and A2780/DDP should be prepared at a concentration of 5 \u0026times;103 cells/100 ul, SKOV3 and SKOV3/DDP at 3.5\u0026times; 103 cells/100 ul, and mix thoroughly and add 100ul to a 96-well plate. The plates were incubated in the incubator for 24 hours. The plates were grouped according to experimental needs, with 3 replicate wells per group. At the end of the experiment, cells were incubated with 10 \u0026micro;L of CCK-8 reagent (sigma Aldrich; Merck KGaA) for 2h at 37\u0026deg;C. The results were detected at 450 nm on a microplate reader (TECAN infinite; TECAN Austria GmbH, groedig, Austria). The percent viability of cells was calculated using the following formula. Cell viability (%) = [A (drug added) -A (blank)]/ [A (0 drug added) -A (blank)] \u0026times; 100%. Each experiment was triplicated independently.\u003c/p\u003e\n\u003ch3\u003ePlane clone formation assay\u003c/h3\u003e\n\u003cp\u003eThe control group, 5umol/LDDP group and 5umol/LDDP\u0026thinsp;+\u0026thinsp;5umol/L Tβ4-17peptide group after 48 hours of intervention were selected for colony formation experiments based on the results of the preliminary CCK8 cell viability assay. The above intervention groups were counted separately and the cells (500 cells/well) were seeded into 6-well plates. After cultured for 14 days, colonies were fixed with 4% paraformaldehyde at room temperature for 20 min and stained with crystal violet for 20 min, and the numbers of visible colonies were counted.\u003c/p\u003e\n\u003ch3\u003eEDU fluorescence proliferation assay\u003c/h3\u003e\n\u003cp\u003eCell configuration concentration was the same as CCK8 assay. The groups were grouped according to the experimental needs and incubated for 2 hours after adding 50ul of EDU working solution. Remove the EDU working solution, add 100ul of 4% paraformaldehyde for fixation for 15-30min, and then incubate with cell permeabilization solution for 10-15min in room temperature environment. The Click reaction solution was prepared according to the instructions and the number of samples to be tested, 50ul was taken and added to the wells to be tested and then incubated for 1 hour. Each well received 100 L of the prepared 1X Hoechst solution and was incubated at room temperature for 20 minutes and photographed under a fluorescence microscope.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eWound healing assay\u003c/h2\u003e\u003cp\u003eCells were cultured to confluence in 6-well plates. A 200 \u0026micro;L pipette tip was used to scratch in each well. Cells were rinsed 3 times with PBS, and then the corresponding culture solution containing the drug to be tested was added separately according to the experimental grouping requirements and placed in the incubator. The 6-well plates were taken out at 0 and 48 hours of drug addition to take images, which were then analyzed by Image J.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eTranswell assays\u003c/h3\u003e\n\u003cp\u003eCells treated in advance with 5\u0026micro;mol/L DDP, 5\u0026micro;mol/L DDP\u0026thinsp;+\u0026thinsp;5\u0026micro;mol/LTβ4\u0026ndash;17 peptide for 48 hours were resuspended in serum-free cell culture medium and counted as 8\u0026times;104 cells, which were categorized into control, DDP, and DDP\u0026thinsp;+\u0026thinsp;Tβ4-17peptide groups. In a 24-well plate, 800ul of culture medium containing 20% FBS was first added, followed by 200ul of serum-free cell suspension evenly dripped into the chamber, and incubated in the incubator. After 24 hours, the culture medium was discarded, fixed for 15\u0026ndash;30 min, and then stained with crystal violet for 20 min. The cells in the lower chamber were observed under the microscope, photographed and counted.\u003c/p\u003e\n\u003ch3\u003eTUNEL assays\u003c/h3\u003e\n\u003cp\u003eCell configuration concentration was the same as CCK8 assay. Treated with 5\u0026micro;mol/L DDP, 5\u0026micro;mol/L DDP\u0026thinsp;+\u0026thinsp;5\u0026micro;mol/L Tβ4\u0026ndash;17 peptide. After 48 h, 4% paraformaldehyde for fixation for 20 min was added and incubated for 5 min at room temperature with potent permeabilization solution. TUNEL assay solution was prepared according to the instructions and the number of experimental samples. 50\u0026micro;l TUNEL assay solution was added to the wells of the samples to be tested and incubated for 1 hour. Then add 100ul DAPI staining solution, incubate for 10min at room temperature. PBS was washed 3 times and photographed under fluorescence microscope observation.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eFlow Cytometry Analysis\u003c/h2\u003e\u003cp\u003eCells were treated with different drugs (0, 5\u0026micro;mol/L DDP, 5\u0026micro;mol/L DDP\u0026thinsp;+\u0026thinsp;5\u0026micro;mol/LTβ4\u0026ndash;17) for 48 h, and trypsin digestion was added. The precipitate was gently resuspended with pre-cooled PBS and counted, and 1\u0026times;105 cells/ml resuspended were taken, centrifuged at 1000 rpm for 5 min, and the cells were resuspended by adding Annexin V-FITC conjugate and gently mixed. Add 10\u0026micro;l of propidium iodide staining solution, incubate for 20min at room temperature, and then test on the machine.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eReverse transcription-quantitative PCR (RT-qPCR)\u003c/h2\u003e\u003cp\u003eThe primers were designed by Primer 6.0 software according to genes sequences searched by Primer-Bank, and synthetized by Shanghai Shenggong Biological Co., Ltd.. Ovarian cancer cells were harvested using RNAkey \u0026trade; Reagent, RNA was reverse-transcribed to cDNA using EvoM-MLV Reverse Transcription Kit (Accurate Biotechnology) following the manufacturer\u0026rsquo;s instructions. The reverse transcription reaction conditions were 37\u0026deg;C for 15min and 85\u0026deg;C for 5sec. Real time PCR reaction conditions were as follows: 95\u0026deg;C pre-denaturation 30 s; 95\u0026deg;C \u0026times;5 s, 60\u0026deg;C\u0026times;30 s (40 cycles). GAPDH was used as the housekeeping gene. For target gene, mRNA expression levels were calculated using the 2‑ΔΔCt method (ΔCt\u0026thinsp;=\u0026thinsp;target gene Ct ‑ GAPDH Ct value). The primer sequence is as follows: GAPDH, 5\u0026rsquo;-GAGTCAACGGATTTGGTCGT-3' and 5\u0026rsquo;-TTGATTTTGGAGGGATCTCG-3'. NF-κB, 5\u0026rsquo;-AATCCAGTGTGTGAAGAAGC-3' and 5\u0026rsquo;- GCTGCTCTTCTATAGGAACT-3'.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eWestern Blot assay\u003c/h2\u003e\u003cp\u003eCells were collected and lysed after 48h of treatment with different drugs. Equal amounts of protein lysate were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to PVDF membranes. 5% skimmed milk was closed for 2 hours and incubated with primary antibodies overnight at 4\u0026deg;C. The primary antibody used in this study are as follows: Anti-β-actin Rabbit Antibody (Abmart, P30002), Anti-NF-κB (P65) Rabbit Antibody (Abmart, T55034). Then, horseradish peroxidase-conjugated secondary antibody (HRP-conjugated goat anti-rabbit IgG). After incubation at 37 \u0026deg; C for 2 h, protein bands were visualized by enhanced chemiluminescence Plus kit (Beyotime, Shanghai, China). Images were acquired by an automated chemiluminescence image analysis system (Tanon, Shanghai, China).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eImage J software was used for cell migration and protein gray value analysis, and GraphPad Prism9.0 performed statistical analysis and mapping of the data. The experimental results of each group were repeated three times. Statistical results All values were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (Means\u0026thinsp;\u0026plusmn;\u0026thinsp;SD), and the comparison of samples between two groups was performed by t-test. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered as a significant difference.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eScreening and expression validation of Tβ4\u0026ndash;17 peptide in cisplatin-resistant/sensitive sera of ovarian cancer\u003c/h2\u003e\u003cp\u003eOur group successfully screened 41 differentially expressed peptide sequences using ITRAQ technology in the early stage\u003csup\u003e(Li 2025)\u003c/sup\u003e .Analysis of the basic characteristics of the differential peptides revealed that the isoelectric points of these peptides were in the range of 3.0\u0026ndash;12.5, but mainly in the acidic range (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The molecular weights of these peptides were mainly in the range of 800\u0026ndash;1600 Da, which was consistent with the principle of peptide identification (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). In order to further understand the physicochemical properties and related information of Tβ4\u0026ndash;17 peptide, we used the ExPASy and ProtParam online programs and discovered: Tβ4\u0026ndash;17 consists of 16 amino acids, with molecular weight of 1,868.09 Da, isoelectric point of 4.86, and instability index of 44.26, which is an unstable polypeptide, the fat-solubility index is 30.63 and the total average hydrophilicity index was \u0026minus;\u0026thinsp;1.581 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This suggests that Tβ4\u0026ndash;17 is a hydrophilic small molecule peptide. We applied the PRM technique to further define the expression of Tβ4\u0026ndash;17 polypeptide in cisplatin-resistant/sensitive sera of ovarian cancer (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The results showed that Tβ4\u0026ndash;17 peptide was significantly under-expressed in cisplatin-resistant serum of ovarian cancer.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePhysicochemical Properties and Related Information of the Tβ4\u0026ndash;17 Peptide.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePhysicochemical Characteristics\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eValue\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNumber of amino acids\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1868.09\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTheoretical PI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e4.86\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eInstability index\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e44.26\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAliphatic index\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e30.63\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGrand average of hydropathicity\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e-1.581\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eRelative expression of target peptides in cisplatin-resistant/sensitive serum of ovarian cancer.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSample\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eR (Drug-resistant serum values)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eS (Sensitive serum values)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e952434\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1287873\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e844393\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1875993\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e804709\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1641410\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e969653\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1940420\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1255356\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1693780\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e983715\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e973706\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1171817\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2095880\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e921588\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1179725\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1573706\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e968543\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1588882\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1724969\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e874026\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1684198\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1418046\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1392530\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eIdentification of Cisplatin-resistant Ovarian Cancer Cells A2780/DDP and SKOV3/DDP\u003c/h2\u003e\u003cp\u003eWe applied the CCK8 assay to detect whether A2780/DDP and SKOV3/DDP are resistant to drugs. The results showed that the number of drug-resistant and parental cells surviving decreased with the increase of DDP concentration. Moreover, the survival rate of parental cells was lower than that of drug-resistant cells at the same concentration of DDP (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, G). According to the absorbance values measured by CCK8, the resistance index were 5.28 and 2.08 for A2780/DDP and 5.06 and 2.51 for SKOV3/DDP at 24 and 48 hours(Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The above results confirmed that both cells were resistant.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eIC50 values and RI values of ovarian cancer parental cells and drug-resistant cells at 24H and 48H.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTime\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eA2780\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eA2780/DDP\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSKOV3\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSKOV3/DDP\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e24H-IC50(\u0026micro;mol/L)\u003c/p\u003e\u003cp\u003e48H-IC50(\u0026micro;mol/L)\u003c/p\u003e\u003cp\u003e24H-RI\u003c/p\u003e\u003cp\u003e48H-RI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.269\u003c/p\u003e\u003cp\u003e4.553\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e27.82\u003c/p\u003e\u003cp\u003e9.512\u003c/p\u003e\u003cp\u003e5.28\u003c/p\u003e\u003cp\u003e2.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6.95\u003c/p\u003e\u003cp\u003e5.64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e35.17\u003c/p\u003e\u003cp\u003e14.17\u003c/p\u003e\u003cp\u003e5.06\u003c/p\u003e\u003cp\u003e2.51\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eTβ4\u0026ndash;17 enhanced DDP-inhibited of human ovarian cancer cell viability and migration\u003c/h2\u003e\u003cp\u003eOur experiments have confirmed that 5\u0026micro;mol/L of DDP is an optimal concentration for the experiment. We tested the effects of different concentrations of Tβ4\u0026ndash;17 (1\u0026micro;mol/L, 5\u0026micro;mol/L, 25\u0026micro;mol/L) combined with DDP on cell proliferation. CCK8 results showed that Tβ4\u0026ndash;17 combined with DDP resulted in a significant dose-dependent decrease in ovarian cancer parental and resistant cell viability compared to DDP alone. And the inhibition of viability in the drug-resistant cells was stronger than that of parental cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-D). Based on this result, we set the concentration of Tβ4\u0026ndash;17 to 5\u0026micro;mol/L for subsequent cell experiments. Then, we carried out plate clone formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eE) and EDU tests (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, G) to confirm the growth inhibition impact of T4-17 on ovarian cancer cells. The results were consistent with the CCK8 assay, with the lowest number of viable cells in the 5\u0026micro;mol/L Tβ4\u0026ndash;17 combined with DDP treatment group. These results indicate that Tβ4\u0026ndash;17 reduces the resistance of ovarian cancer cells to DDP.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo explore whether Tβ4\u0026ndash;17 combined with DDP caused migration inhibition. We first examined cell migration rates using a cell scratch assay, which showed that Tβ4\u0026ndash;17 combined with DDP treatment slowed cell convergence to closure (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eH, I). This migration inhibition was stronger for ovarian cancer resistant cells. Subsequently, we used transwell assay to detect cell migration after co-treatment. When Tβ4\u0026ndash;17 was coupled with DDP therapy, the number of cells passing through the microtiter wells was decreased after 48 hours compared to the DDP treatment group alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ). This suggests that Tβ4\u0026ndash;17 enhances the migration inhibition of ovarian cancer cells by DDP.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eTβ4\u0026ndash;17 increased DDP-induced apoptosis in human ovarian cancer cells\u003c/h2\u003e\u003cp\u003eWe used the TUNEL technique to detect apoptosis in drug-resistant cells in ovarian cancer to investigate the effect of Tβ4\u0026ndash;17 coupled with DDP on apoptosis. The results showed that the number of apoptotic cells was significantly increased in the Tβ4\u0026ndash;17 combined with DDP group compared with the DDP alone group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). Flow cytometry to further analyze revealed that the apoptosis rates of DDP alone treated A2780/DDP and SKOV3/DDP cells were 25.3% and 24.5%, respectively, whereas the apoptosis rates after the addition of Tβ4\u0026ndash;17 were 56.2% and 51.8%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, D). The result suggests that Tβ4\u0026ndash;17 enhances apoptosis induction by DDP.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eTβ4\u0026ndash;17 regulated NF-κB expression\u003c/h2\u003e\u003cp\u003eTo investigate whether NF-κB expression levels are associated with drug resistance, we examined NF-κB expression in ovarian cancer parental cells and drug-resistant cells by qRT-PCR. The results showed that the expression level of NF-κB mRNA in drug-resistant cells was significantly higher than that in parental cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Western blot results showed that NF-κB p65 protein was significantly overexpressed in ovarian cancer resistant cells compared to ovarian cancer parental cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Our result indicates that high expression of NF-κB p65 protein is associated with drug resistance in ovarian cancer.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eGiven the involvement of NF-κB in ovarian cancer drug resistance, we examined the expression level of NF-κB p65 protein in ovarian cancer cells after Tβ4\u0026ndash;17 combined with DDP. Western blot results showed that the NF-κB p65 protein content was decreased after Tβ4\u0026ndash;17 combined with DDP acted on the cells compared to DDP treatment alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, D). It suggests that Tβ4\u0026ndash;17 combined with DDP can downregulate the activation of the NF-κB pathway, which partially reversed cellular resistance to drug.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eTβ4\u0026ndash;17 enhances chemosensitivity of ovarian cancer cells to DDP through the NF-κB pathway\u003c/h2\u003e\u003cp\u003eTo further investigate whether Tβ4\u0026ndash;17 regulates ovarian cancer cells through the NF-κB signaling pathway, PDTC was selected as an NF-κB inhibitor. Western blot analysis revealed that PDTC significantly inhibited the expression of the NF-B p65 protein in drug-resistant ovarian cancer cells. Compared with the decrease in NF-κB p65 protein expression induced by the DDP\u0026thinsp;+\u0026thinsp;PDTC group, interestingly, it furthermore decrease after the addition of Tβ4\u0026ndash;17, suggesting that Tβ4\u0026ndash;17 can effectively down-regulate NF-κB p65 protein expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). We also tested the effect on cell proliferation after application of PDTC. The results of CCK8 assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) and EDU fluorescence assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, D) showed that the PDTC group alone inhibited cell proliferation, suggesting that inhibition of NF-κB expression in the cells slowed down cell growth. The highest cell growth inhibition rate was achieved with the addition of Tβ4\u0026ndash;17 compared to the DDP combined with PDTC group. In addition, we examined the migratory capacity of the cells. The results showed that the cell migration inhibition effect was most obvious in the group treated with the addition of Tβ4\u0026ndash;17 (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, F). Taken together, these results suggest that Tβ4\u0026ndash;17 inhibits the proliferation and migration of drug-resistant cells of ovarian cancer by down-regulating the expression of NF-κB.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo further confirm the relationship between Tβ4\u0026ndash;17 and the NF-κB pathway, TNF-α was used as an activator of NF-κB. Western blot results showed that TNF-α could effectively activate the expression of NF-κB p65 protein in drug-resistant cells of ovarian cancer. NF-κB p65 protein was significantly decreased after application of Tβ4\u0026ndash;17 compared to the DDP\u0026thinsp;+\u0026thinsp;TNF-α group (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). It is suggested that Tβ4\u0026ndash;17 peptide can effectively reverse the expression of NF-κB p65 protein after application of TNF-α. We also analyzed the effect on cell proliferation after application of TNF-α. The results of CCK8 assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003eB) and EDU fluorescence assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, D) showed that TNF-α alone had no significant effect on cell proliferation. The proliferative capacity of the cells in the DDP\u0026thinsp;+\u0026thinsp;TNF-α group was significantly decreased, but the most significant decrease was observed with the addition of Tβ4\u0026ndash;17 peptide. In addition, the results of cell scratch assay showed that TNF-α had no significant effect on cell migration. The ability of cells in the DDP\u0026thinsp;+\u0026thinsp;TNF-α group to aggregate toward the middle was slowed, but the number of resistant cells moving toward the middle was significantly reduced by the addition of Tβ4\u0026ndash;17 peptide (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, F). Taken together, Tβ4\u0026ndash;17 peptide can inhibit cell proliferation and migration through down-regulation of the NF-κB pathway, which in turn enhances the chemo-sensitivity of cells to DDP.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOvarian cancer remains one of the deadliest malignancies among gynecologic cancers due to the lack of early symptoms and the difficulty of clinical diagnosis\u003csup\u003e(Ayhan, et al. 2023)\u003c/sup\u003e. DDP-based platinum-based drugs are commonly used chemotherapeutic agents for the treatment of ovarian cancer. However, long-term and high-dose administration of DDP can lead to serious side effects\u003csup\u003e(Zangouei and Moghbeli 2021)\u003c/sup\u003e. Importantly, the development of drug resistance is frequently observed in patients with ovarian cancer, making DDP less effective and leading to failure of tumor suppression and recurrence\u003csup\u003e(Dall'Acqua, et al. 2017)\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003ePeptides provide us with valuable information, and their expression can be detected throughout the body in organs and tissues as well as in cells and body fluids\u003csup\u003e(Ghose, et al. 2022,Tammen, et al. 2007)\u003c/sup\u003e. Several studies have confirmed that peptides can be used to diagnose or treat various diseases\u003csup\u003e(Guo, et al. 2023)\u003c/sup\u003e. Pan et al\u003csup\u003e(Pan, et al. 2022)\u003c/sup\u003e found that the endogenous peptide PDHPS1 disrupts YAP signaling by interacting with PTPA in ovarian cancer, thereby inhibiting the growth of ovarian cancer cells in vitro and in vivo. In addition, peptides also play an active role in chemosensitivity. GTGKT peptide reduces the angiogenic, tumorigenic and metastatic potential of anticancer-resistant cancer cells and enhances the apoptotic effect and sensitivity of anticancer drugs by inhibiting the binding of tumor-associated genes to the cyclin D1 promoter sequence \u003csup\u003e(Kim, et al. 2017)\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn view of the important role played by peptides in tumor therapy, our group used ITRAQ technology to screen for differentially expressed bioactive peptides from cisplatin-resistant/sensitive sera of ovarian cancer\u003csup\u003e(Li 2025)\u003c/sup\u003e. Among them, the relatively low expression of Tβ4\u0026ndash;17 in drug-resistant sera of ovarian cancer and its possible association with drug resistance. The findings of this study indicate that Tβ4\u0026ndash;17 enhances the cytotoxicity of DDP against ovarian cancer cells, inhibits migration, promotes apoptosis, and increases sensitivity of ovarian cancer cells to cisplatin. Due to the significantly higher cytotoxic effects of Tβ4\u0026ndash;17 and DDP on drug-resistant ovarian cancer cells compared to sensitive cells, it indicates that Tβ4\u0026ndash;17 can synergistically enhance the effects of DDP. Our results also suggest that Tβ4\u0026ndash;17 may enhance the sensitivity of ovarian cancer cells to DDP through the NF-κB signaling pathway, but the related mechanisms require further investigation.\u003c/p\u003e\u003cp\u003eThe drug resistance mechanism of ovarian cancer is highly complex, involving multiple genes and pathways\u003csup\u003e(Freimund, et al. 2018)\u003c/sup\u003e. In recent years, the role of the NF-κB signaling pathway in drug resistance in ovarian cancer has drawn extensive attention. In most cell types, NF-κB exists in an inactive state in the cytoplasm and forms a trimeric complex with the inhibitory factor IκB, thus transcriptionally inactive before the cell is activated by relevant stimuli\u003csup\u003e(Guan, et al. 2022)\u003c/sup\u003e. Activation of the NF-κB pathway has been reported in various types of tumors, leading to enhanced expression of anti-apoptotic proteins, increased secretion of pro-inflammatory cytokines, and promotion of MDR\u003csup\u003e(Yuan, et al. 2020,Tan, et al. 2019)\u003c/sup\u003e. Most chemotherapeutic agents induce some form of DNA damage, causing activation of the NF-κB signalling pathway. Yan et al\u003csup\u003e(Yan, et al. 2014)\u003c/sup\u003e found that activation of the ATM/NF-κB pathway increased chemoresistance in lung cancer cells and enhanced the expression of Bcl-2, Mcl-1, Bcl-xl and ABCG2. Hu et al\u003csup\u003e(Hu, et al. 2021)\u003c/sup\u003e demonstrated that NF-κB activation was associated with glioma drug resistance, leading to temozolomide resistance. Conversely, interfering with NF-κB activity enhances the susceptibility of transplanted tumours to drug resistance, resulting in a reduction in tumour size and weight\u003csup\u003e(Liu, et al. 2021)\u003c/sup\u003e. Overexpression of cytosolic NF-κB in ovarian cancer has been reported to be associated with advanced FIGO staging, residual lesions\u0026thinsp;\u0026ge;\u0026thinsp;1 cm, low histological grade, chemotherapy resistance as well as platinum resistance and refractory disease, as well as a more aggressive ovarian cancer phenotype \u003csup\u003e(Kan, et al. 2020)\u003c/sup\u003e cells. In this study, we observed increased transcriptional activity of NF-kB in drug-resistant ovarian cancer cells compared to parental cells. This result is consistent with previous findings in ovarian cancer cells\u003csup\u003e(Yan, et al. 2017)\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003ePDTC acts as an inhibitor of NF-κB and effectively inhibits its expression. Similarly, in our experiments, PDTC reduced the expression of NF-κB in drug-resistant cells of ovarian cancer, and cell viability was significantly inhibited. Importantly, the addition of Tβ4\u0026ndash;17 co-treatment significantly enhanced the inhibitory effect of cisplatin. In contrast, TNF-α as an NF-κB pathway activator activated NF-κB expression in cells but had no significant effect on cell viability. Previous studies have reported that TNF-α co-administered with PTX enhances the latter's apoptotic response, and its cytotoxicity can be increased in combination with DDP \u003csup\u003e(Noonan, et al. 2020)\u003c/sup\u003e. We demonstrated that TNF-α enhanced the cytotoxicity of DDP, but this cytotoxic effect reached its maximum with the addition of Tβ4\u0026ndash;17.\u003c/p\u003e\u003cp\u003eTaken together, we demonstrated that Tβ4\u0026ndash;17 enhanced the sensitivity of human ovarian cancer cell lines (A2780, SKOV3) to their DDP-resistant cell lines (A2780/DDP, SKOV3/DDP). In addition, NF-κB was shown to be associated with resistance to cisplatin chemotherapy in ovarian cancer. In this study, we verified the alteration of NF-κB protein expression by Tβ4\u0026ndash;17 by applying NF-κB inhibitor (PDTC) and NF-κB activator (TNF-α), respectively. It is speculated that Tβ4\u0026ndash;17 may increase the sensitivity of ovarian cancer cells to DDP by down-regulating the NF-κB signalling pathway. These data help to understand the role of Tβ4\u0026ndash;17 in chemotherapy and may contribute to the development of an effective therapeutic strategy to overcome DDP resistance in ovarian cancer.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e Not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u003c/strong\u003e Not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e The authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003cstrong\u003e：\u003c/strong\u003eThe study was supported by the Harbin Medical University Cancer Hospital Haiyan Research Foundation [grant number JJZD2022-01]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' Contributions\u003c/strong\u003e\u003cstrong\u003e：\u003c/strong\u003eLing Guo wrote the main manuscript text and performed experiments with the assistance of Jing Wang and Nana Li; Yu Ming collected serum samples; Yingxu Li and Peihua Yan performed the statistical analysis; Yajuan Su and Lichen Teng corrected and reviewed the article before submission.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eMarkman, M. 2007. \u0026quot;Development of an ovarian cancer symptom index: possibilities for earlier detection.\u0026quot; Cancer, 110 (1), 226; author reply 227.\u003c/li\u003e\n \u003cli\u003eWang, Y., Ren, S., Jiang, W., Lu, J., Zhang, X., Li, X. et al. 2021. \u0026quot;CA125-Tn ELISA assay improves specificity of pre-operative diagnosis of ovarian cancer among patients with elevated serum CA125 levels.\u0026quot; ANN TRANSL MED, 9 (9), 788.\u003c/li\u003e\n \u003cli\u003eBristow, R.E., Montz, F.J., Lagasse, L.D., Leuchter, R.S. and Karlan, B.Y. 1999. \u0026quot;Survival impact of surgical cytoreduction in stage IV epithelial ovarian cancer.\u0026quot; Gynecol Oncol, 72 (3), 278-287.\u003c/li\u003e\n \u003cli\u003eLedermann, J., Raja, F., Fotopoulou, C., Gonzalez-Martin, A., Colombo, N. and Sessa, C. 2018. \u0026quot;Newly diagnosed and relapsed epithelial ovarian carcinoma: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up.\u0026quot; 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Cancer science.108 (7), 1405-1413.\u003c/li\u003e\n \u003cli\u003eNoonan, A., Cousins, A., Anderson, D., Zeligs, K., Bunch, K., Hernandez, L. et al. 2020. \u0026quot;Matrix Drug Screen Identifies Synergistic Drug Combinations to Augment SMAC Mimetic Activity in Ovarian Cancer.\u0026quot; Cancers.12 (12).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"medical-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"medo","sideBox":"Learn more about [Medical Oncology](https://www.springer.com/journal/12032)","snPcode":"12032","submissionUrl":"https://submission.nature.com/new-submission/12032/3","title":"Medical Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"ovarian cancer1, peptides2, Tβ4-173, DDP4, NF-κB5","lastPublishedDoi":"10.21203/rs.3.rs-6951674/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6951674/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOvarian cancer is a gynecologic malignancy with high mortality and poor prognosis. Chemoresistance is a key cause of ovarian cancer recurrence and metastasis. It has been found that some bioactive peptides can inhibit the growth and metastasis of cancer cells and promote cell apoptosis, thus exerting anti-cancer effects. Tβ4-17 is a small polypeptide that we selected using ITRAQ technology, and its precursor protein is thymosin β4. This study mainly investigated its effect in combination with cisplatin (DDP) on the proliferation, migration and apoptosis of ovarian cancer resistant cells and related molecular mechanisms. Our results showed that Tβ4-17 peptide combined with DDP significantly inhibited the proliferation and migration of drug-resistant cells in ovarian cancer, promoted apoptosis, and increased the chemosensitivity of ovarian cancer cells to DDP. In addition, qRT-PCR and Western blot showed that NF-κB was significantly highly expressed in cisplatin-resistant cells of ovarian cancer. After application of NF-κB inhibitors and activators, Western blot, CCK8, EDU fluorescence proliferation assay, and cell scratch assay showed that Tβ4-17 peptide down-regulated NF-κB p65 protein expression and inhibited cell proliferation and migration. In conclusion, our study demonstrates that Tβ4-17 peptide enhances the sensitivity of ovarian cancer cells to DDP by down-regulating NF-κB expression\u003c/p\u003e","manuscriptTitle":"Tβ4-17 peptide enhances the chemo-sensitivity of ovarian cancer cells to DDP by affecting NF-κB signaling pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-23 15:30:27","doi":"10.21203/rs.3.rs-6951674/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-23T18:24:23+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-20T13:02:22+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-12T22:11:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"339511335985744244295238183273624769962","date":"2025-09-06T09:16:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"54228239342676718992043544392355393821","date":"2025-09-02T16:03:32+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-06T02:15:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"172668098040444211483566019487769000378","date":"2025-07-21T17:56:53+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-21T02:34:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-23T02:56:10+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-23T02:55:40+00:00","index":"","fulltext":""},{"type":"submitted","content":"Medical Oncology","date":"2025-06-23T00:31:26+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"medical-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"medo","sideBox":"Learn more about [Medical Oncology](https://www.springer.com/journal/12032)","snPcode":"12032","submissionUrl":"https://submission.nature.com/new-submission/12032/3","title":"Medical Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"02375065-9812-4077-9191-912c7a41e01a","owner":[],"postedDate":"July 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-10T15:59:22+00:00","versionOfRecord":{"articleIdentity":"rs-6951674","link":"https://doi.org/10.1007/s12032-025-03106-4","journal":{"identity":"medical-oncology","isVorOnly":false,"title":"Medical Oncology"},"publishedOn":"2025-11-08 15:57:04","publishedOnDateReadable":"November 8th, 2025"},"versionCreatedAt":"2025-07-23 15:30:27","video":"","vorDoi":"10.1007/s12032-025-03106-4","vorDoiUrl":"https://doi.org/10.1007/s12032-025-03106-4","workflowStages":[]},"version":"v1","identity":"rs-6951674","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6951674","identity":"rs-6951674","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}