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

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 resistance cells in ovarian cancer, promoted apoptosis, and increased the chemo-sensitivity of ovarian cancer cells to DDP. In addition, qRT-PCR and Western blot showed that NF-κB was significantly highly expressed in DDP-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.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12032-025-03106-4.

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 [1, 2].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 [3, 4]. 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 [5, 6]. 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 [7]. 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 [8]. 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 [9–11]. Importantly, some peptides can synergize the killing effect of chemotherapeutic drugs on cells and enhance chemo-sensitivity [12, 13]. For example, the peptide Mastoparan combined with gemcitabine significantly inhibited the growth of a mouse model of breast cancer after treatment [14]. 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 DDP-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 [15, 16]. We hypothesized that Tβ4–17 would influence drug 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

Clinical data

From October 2020 to September 2021, serum samples were collected from 17 DDP-resistant (experimental group) and 17 DDP-sensitive (control group) ovarian cancer patients treated at Harbin Medical University Cancer Hospital. Within each group, five samples were randomly selected for differential peptide screening, and the remaining 12 were used for targeted quantification of candidate peptides. Patients with concurrent diseases or other malignancies were excluded. This study involved the secondary use of clinically archived serum specimens; exemption from informed consent was approved by the Ethics Committee of Harbin Medical University Cancer Hospital.

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 (100 mg) 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. DDP-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 5 A (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

Prepare A2780 and A2780/DDP at 5 × 10³ cells/100 µL and SKOV3 and SKOV3/DDP at 3.5 × 10³ cells/100 µL; mix each suspension thoroughly and dispense 100 µL into a 96-well plate. Incubate the plates for 24 h, then assign wells to experimental groups (three replicates per group). At the end of treatment, add 10 µL CCK-8 reagent (Sigma-Aldrich; Merck KGaA) and incubate for 2 h at 37 °C. Measure absorbance at 450 nm with a microplate reader (Tecan Infinite; Tecan Austria GmbH, Grödig, Austria). Calculate cell viability (%) as:

[A(drug) − A(blank)]/[A(0 drug) − A(blank)] × 100%.

Each experiment was performed in triplicate.

Plane clone formation assay

Based on the preliminary CCK-8 viability screen, cells that had received 48 h of either control treatment, 5 µmol/LDDP alone, or 5 µmol/LDDP plus 5 µmol/L Tβ4–17 peptide were selected for clonogenic assays. Each group was trypsinized, counted, and seeded at 500 cells per well in 6-well plates. After 14 days of culture, colonies were fixed with 4% paraformaldehyde for 20 min at room temperature, stained with 0.5% crystal violet for 20 min, rinsed, air-dried, and colonies ≥ 50 cells were enumerated manually under a light microscope.

EDU fluorescence proliferation assay

Cells were seeded at the same density used for the CCK-8 assay and grouped according to the experimental design. After 50 µL of EdU working solution was added, the plates were incubated for 2 h. The EdU solution was then removed, and the cells were fixed with 100 µL of 4% paraformaldehyde for 15–30 min, followed by permeabilization with 100 µL of permeabilization buffer for 10–15 min at room temperature. The Click reaction cocktail was prepared fresh according to the manufacturer’s instructions; 50 µL was added to each well and incubated for 1 h in the dark. Finally, 100 µL of 1× Hoechst 33,342 was added per well, incubated for 20 min at room temperature, and images were acquired on a fluorescence microscope.

Wound healing assay

A2780 and A2780/DDP cells were prepared at 3 × 10⁵ cells/ml, while SKOV3 and SKOV3/DDP cells were adjusted to 1.5 × 10⁵ cells/ml; 1 ml of each suspension was seeded into separate wells of a six-well plate. After overnight incubation, the cultures were grown to confluence before a standardized scratch wound was generated. Create a uniform scratch with a 200-µL pipette tip, rinse three times with PBS, then replace with serum-free medium containing the indicated drugs according to experimental groups. Immediately record the 0-h image; return plates to a 37 °C, 5% CO₂ incubator. Re-image identical fields at 48 h.The same field was imaged again at 48 h. Wound closure was quantified using ImageJ software. All experiments were performed in triplicate.

Transwell assays

Cells that had been pre-treated for 48 h with 5 µmol/L DDP alone or with 5 µmol/L DDP plus 5 µmol/L Tβ4–17 peptide were harvested, resuspended in serum-free medium, and adjusted to a density equivalent to 8 × 10⁴ cells. Three experimental cohorts were established: control, DDP, and DDP + Tβ4–17 peptide. For the migration assay, 800 µL of medium supplemented with 20% FBS was first placed in each well of a 24-well plate, after which 200 µL of the serum-free cell suspension was carefully layered into the upper chamber. Following 24 h of incubation, the medium was removed, and the cells were fixed for 15–30 min and subsequently stained with crystal violet for 20 min. Cells that had migrated to the lower surface of the membrane were visualized under a microscope, imaged, and enumerated.

TUNEL assays

Cells were seeded at the same density employed in the CCK-8 assay and exposed to 5 µmol/L DDP or 5 µmol/L DDP plus 5 µmol/L Tβ4–17 peptide. After 48 h, cultures were fixed with 4% paraformaldehyde for 20 min, permeabilised with potent permeabilisation solution for 5 min at room temperature, and processed for TUNEL staining. The TUNEL reaction cocktail was prepared strictly according to the manufacturer’s instructions in proportion to the number of specimens; 50 µL of the mixture was applied to each well and incubated for 1 h in the dark. Nuclei were counterstained with 100 µL DAPI for 10 min at room temperature, washed three times with PBS, and visualised under a fluorescence microscope for image acquisition.

Flow cytometry analysis

Cells were exposed to the indicated treatments—vehicle, 5 µmol/L DDP, or 5 µmol/L DDP plus µ5 mol/L Tβ4–17—for 48 h, harvested by trypsinization, and washed once with ice-cold PBS. After counting, 1 × 10⁵ cells were pelleted at 1 000 rpm for 5 min, resuspended in Annexin V-FITC binding buffer containing the conjugated antibody, and gently mixed. Ten microlitres of propidium iodide staining solution was added, and the samples were incubated for 20 min at room temperature in the dark before flow-cytometric analysis.

Reverse transcription-quantitative PCR (RT-qPCR)

Primer pairs were designed with Primer 6.0 on the basis of sequences obtained from Primer-Bank and were commercially synthesized by Shanghai Shenggong Biological Co., Ltd. Total RNA was isolated from ovarian-cancer cells with RNAkey™ Reagent and reverse-transcribed with the EvoM-MLV Reverse Transcription Kit (Accurate Biotechnology) according to the manufacturer’s instructions (37 °C for 15 min, 85 °C for 5 s). Quantitative real-time PCR was performed under the following conditions: 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. GAPDH served as the internal reference, and relative mRNA expression was calculated with the 2-ΔΔCt method (ΔCt = Ct target − Ct GAPDH). The primer sequences are as follows: GAPDH, 5′-GAGTCAACGGATTTGGTCGT-3′ and 5′-TTGATTTTGGAGGGATCTCG-3′; NF-κB, 5′-AATCCAGTGTGTGAAGAAGC-3′ and 5′-GCTGCTCTTCTATAGGAACT-3′.

Western blot assay

After 48 h of drug treatment, cells were harvested and lysed. Equal amounts of protein were separated by 10% SDS–PAGE and electro-transferred to PVDF membranes. The membranes were blocked with 5% skimmed milk for 2 h at room temperature and then incubated overnight at 4 °C with primary antibodies. Subsequently, the membranes were probed with horseradish peroxidase-conjugated secondary antibody for 2 h at 37 °C. Protein bands were visualized using an enhanced chemiluminescence Plus kit (Beyotime, Shanghai, China) and imaged with an automated chemiluminescence system (Tanon, Shanghai, China).

Statistical analysis

Cell-migration areas and band intensities were quantified with ImageJ; statistical analyses and graphics were prepared in GraphPad Prism 9.0. All experiments were performed in triplicate. Data are presented as mean ± SD. Comparisons between two groups were evaluated by two-tailed Student’s t-test, and P < 0.05 was considered statistically significant.

Results

Screening and expression validation of Tβ4–17 peptide in DDP-resistant/sensitive Sera of ovarian cancer

Our group successfully screened 41 differentially expressed peptide sequences using ITRAQ technology in the early stage [17].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. 1A). 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. 1B). 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.Using PRM—a highly specific targeted protein quantification technique—we further determined the expression of the Tβ4–17 polypeptide in DDP-resistant versus DDP-sensitive ovarian cancer sera (Table 2; Fig. 1C). The results showed that the Tβ4–17 peptide was significantly under-expressed in the DDP-resistant serum samples.

Fig. 1.

Screening and identification of Tβ4–17 and detection of drug resistance cells. (A) Isoelectric Point Distribution of Peptides. (B) Molecular weight distribution of peptides. (C) Tβ4–17 expression in DDP-resistant/sensitive serum of ovarian cancer detected by PRM technique. (D, E) CCK8 detects A2780/DDP resistance at 24 H and 48 H. (F, G) CCK8 detects SKOV3/DDP resistance at 24 H and 48 H. The data were presented as the mean ± SD (n = 3), *P < 0.05, t test

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 DDP-resistant/sensitive serum of ovarian cancer

Sample	R (drug resistance serum values)	S (Sensitive serum values)
1	952,434	1,287,873
2	844,393	1,875,993
3	804,709	1,641,410
4	969,653	1,940,420
5	1,255,356	1,693,780
6	983,715	973,706
7	1,171,817	2,095,880
8	921,588	1,179,725
9	1,573,706	968,543
10	1,588,882	1,724,969
11	874,026	1,684,198
12	1,418,046	1,392,530

Identification of DDP-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 resistance and parental cells surviving decreased with the increase of DDP concentration. Moreover, the survival rate of parental cells was lower than that of drug resistance cells at the same concentration of DDP (Fig. 1D, 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 h(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 resistance cells at 24 H and 48 H

Time	A2780	A2780/DDP	SKOV3	SKOV3/DDP
24 H-IC50(µmol/L) 48 H-IC50(µmol/L) 24 H-RI 48 H-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 resistance cells was stronger than that of parental cells (Fig. 2A). 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. 2E) and EDU tests (Fig. 2F, 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.

Fig. 2.

Tβ4–17 enhanced DDP inhibition of human ovarian cancer cell viability and migration. (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), *P < 0.05, **P < 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), *P < 0.05, **P < 0.01, t test

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. 2H, 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 h compared to the DDP treatment group alone (Fig. 2J). 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 resistance 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. 3A, 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. 3C, D). The result suggests that Tβ4–17 enhances apoptosis induction by DDP.

Fig. 3.

Tβ4–17 increased DDP-induced apoptosis inhuman ovarian cancer cells. (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. The data were presented as the mean ± SD (n = 3), *P < 0.05, t test. (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), *P < 0.05, t test

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 resistance cells by qRT-PCR. The results showed that the expression level of NF-κB mRNA in drug resistance cells was significantly higher than that in parental cells (Fig. 4A). Western blot results showed that NF-κB p65 protein was significantly overexpressed in ovarian cancer resistant cells compared to ovarian cancer parental cells (Fig. 4B). Our result indicates that high expression of NF-κB p65 protein is associated with drug resistance in ovarian cancer.

Fig. 4.

Tβ4–17 regulated NF-κB expression. (A) NF-κB mRNA expression was analyzed using qRT-PCR. (B) Western-blot images and relative expression levels of NF-κB in A2780, A2780/DDP, SKOV3 and SKOV3/DDP cells. Proteins were detected by Western blot with β-actin as internal reference.(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), *P < 0.05, **P < 0.01, t test

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. 4C, 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 chemo-sensitivity 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 resistance 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. 5A). We also tested the effect on cell proliferation after application of PDTC. The results of CCK8 assay (Fig. 5B) and EDU fluorescence assay (Fig. 5C, 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. 5E, F). Taken together, these results suggest that Tβ4–17 inhibits the proliferation and migration of drug resistance cells of ovarian cancer by down-regulating the expression of NF-κB.

Fig. 5.

Effect of Tβ4–17 on drug resistance cells of ovarian cancer after application of PDTC. (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), *P < 0.05, **P < 0.01, ***P < 0.001, t test

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 resistance cells of ovarian cancer. NF-κB p65 protein was significantly decreased after application of Tβ4–17 compared to the DDP + TNF-α group (Fig. 6A). 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. 6B) and EDU fluorescence assay (Fig. 6C, 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. 6E, 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.

Fig. 6.

Effect of Tβ4–17 on drug resistance cells of ovarian cancer after application of TNF-α. (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, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, t test

Discussion

Ovarian cancer remains the most lethal gynaecological malignancy because early-stage disease is asymptomatic and there are no reliable tools for early detection [18]. Although platinum-based regimens containing cisplatin constitute first-line chemotherapy, cumulative and high-dose exposure is associated with severe systemic toxicity [19]. Moreover, acquired resistance inevitably emerges in a substantial proportion of patients, abrogating cisplatin efficacy and precipitating tumour progression and relapse[20].

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 [21, 22]. Several studies have confirmed that peptides can be used to diagnose or treat various diseases [23]. Pan et al. [24]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.MOTS-c, a mitochondria-derived peptide, dose-dependently suppresses the proliferation, migration, and invasion of ovarian cancer cells while concurrently inducing cell-cycle arrest and apoptosis. These anti-tumour effects are mediated through attenuation of USP7-dependent deubiquitination of LARS1 and the resultant proteasomal degradation of the latter [25]. In addition, peptides also play an active role in chemo-sensitivity. 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 [26].Lacroix, A. et al. [27] identified a novel tumour-penetrating peptide, PEP-010, that exerts pro-apoptotic activity by selectively disrupting the interaction between protein phosphatase 2 A (PP2A) and caspase-9. PP2A negatively regulates and inactivates numerous apoptosis- and survival-associated downstream targets—including Bcl-2 family proteins and Akt—and PEP-010 effectively triggers apoptosis in multiple chemoresistant ovarian cancer cell lines [28].Peptides also modulate tumour progression by reshaping the tumour microenvironment. Boylan et al. [29] demonstrated that the peptide N4-P10 disrupts cell aggregation in ovarian-cancer ascites and potentiates cisplatin cytotoxicity. Beyond ovarian cancer, peptides are emerging as key players in oncotherapy and drug-delivery systems: the peptide–drug conjugate MEL-dKLA re-programmes M2-like tumour-associated macrophages, thereby suppressing prostate-tumour growth [30], whereas the circular peptide circZKSaa impedes hepatocellular carcinoma (HCC) progression and sensitises HCC cells to sorafenib [31].

Given the pivotal role of bioactive peptides in tumor therapy, our group employed iTRAQ to screen for differentially expressed peptides in sera derived from DDPn-resistant/sensitive ovarian cancer patients [17]. Among the candidates, thymosin β4-derived peptide Tβ4–17 was significantly down-regulated in resistant sera, suggesting a potential association with chemoresistance. The present study demonstrates that exogenous Tβ4–17 augments DDP cytotoxicity against ovarian cancer cells, attenuates migration, facilitates apoptosis, and restores DDP sensitivity. Notably, the combined treatment of Tβ4–17 and DDP elicited a disproportionately greater cytotoxic effect in resistant cells than in their sensitive counterparts, indicating a synergistic interaction. Mechanistically, our data implicate the NF-κB signaling axis as a putative mediator of Tβ4–17–induced chemosensitization; however, the precise molecular underpinnings require further investigation.

Ovarian-cancer chemoresistance is orchestrated by a polygenic network that engages multiple signaling hubs [32]. 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 heterotrimers are sequestered in the cytosol through high-affinity binding to IκB inhibitory proteins, ensuring transcriptional silence until extracellular or genotoxic stimuli trigger ubiquitin-mediated IκB degradation [33]. Constitutive NF-κB activation is now documented across a spectrum of solid tumours, where it transcriptionally up-regulates anti-apoptotic cohorts (Bcl-2, Mcl-1, Bcl-xL), pro-inflammatory cytokines and ATP-binding-cassette efflux pumps, thereby collectively driving multidrug resistance (MDR) [34, 35]. Indeed, most front-line genotoxic agents provoke double-strand breaks that initiate ATM-dependent NF-κB signaling; Yan et al. demonstrated that this ATM–NF-κB circuitry augments expression of Bcl-2, Mcl-1, Bcl-xL and ABCG2, conferring cisplatin resistance in non-small-cell lung cancer [36]. Similarly, Hu et al. linked NF-κB hyper-activation to temozolomide refractoriness in glioblastoma, whereas genetic or pharmacologic NF-κB suppression restored chemosensitivity and reduced tumour burden in xenograft models [37, 38]. In high-grade serous ovarian carcinoma, cytoplasmic NF-κB over-expression correlates with advanced FIGO stage (III/IV), sub-optimal cytoreduction (residual ≥ 1 cm), low histological grade, and both primary platinum resistance and refractory relapse, collectively defining an aggressive disease phenotype [39].Consistent with these data, we now report a significant increase in NF-κB transcriptional activity in DDP-resistant ovarian-cancer cells relative to their drug-naïve parental counterparts, corroborating earlier observations in this malignancy [40].

PDTC is a well-characterized NF-κB inhibitor that suppresses both nuclear translocation and DNA-binding activity of the transcription factor. Consistently, we observed that PDTC down-regulated total and nuclear NF-κB p65 in DDP-resistant ovarian-cancer cells and markedly reduced cell viability. Importantly, concomitant administration of Tβ4–17 significantly potentiated the growth-inhibitory effect of cisplatin, indicating a synergistic interaction. Conversely, TNF-α, a canonical NF-κB agonist, increased NF-κB transcriptional activity without appreciably altering basal viability. Although previous work has shown that TNF-α priming augments paclitaxel-induced apoptosis and enhances cisplatin cytotoxicity [41], we demonstrate that the maximal cytocidal effect of cisplatin is achieved only when TNF-α is combined with Tβ4–17, underscoring the peptide’s unique capacity to override NF-κB–mediated chemoresistance.

Collectively, we demonstrate that Tβ4–17 restores cisplatin sensitivity in both parental (A2780, SKOV3) and corresponding DDP-resistant (A2780/DDP, SKOV3/DDP) human ovarian-cancer cell lines. Pharmacologic interrogation with the NF-κB inhibitor PDTC and the agonist TNF-α further reveals that Tβ4–17 negatively regulates NF-κB signaling, and its chemosensitizing effect is abolished when NF-κB is constitutively activated. These findings indicate that Tβ4–17 overcomes platinum resistance, at least in part, by down-regulating the NF-κB pathway. Our data provide mechanistic insight into the peptide’s activity and support its further development as a rational adjunct to re-sensitize ovarian tumours to cisplatin-based therapy.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Ling Guo and Haibing Wang 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.#Ling Guo and Haibing Wang contributed equally to this work.#Yajuan Su and Lichen Teng are co-corresponding authors.

Funding

The study was supported by the Harbin Medical University Cancer Hospital Haiyan Research Foundation [grant number JJZD2022-01].

Data availability

All data are available upon request to the corresponding authors.

Declarations

Competing interests

The authors declare no competing interests.

Consent for publication

Not applicable.

Ethics statement

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Human ethics and consent to participate statement

This study involved the secondary use of clinically archived serum specimens; exemption from informed consent was approved by the Ethics Committee of Harbin Medical University Cancer Hospital.

Limitations and future directions

Due to limited experimental resources, some assays could not be undertaken; we will complete the necessary in vivo studies once our research facilities are upgraded.