The human OC cell lines OV-90 and SK-OV-3, and normal ovarian epithelial IOSE80 cells were purchased form Zhong Qiao Xin Zhou Biotechnology Co., Ltd. Cisplatin-resistant SK-OV-3 cells (SK-OV-3-R) were obtained from Zhejiang Meisen Cell Technology Co Ltd. All cells were maintained in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin (Beijing Solarbio Science & Technology Co., Ltd.), and cultured at 37˚C in a humidified atmosphere containing 5% CO₂. Gramine (HY-N0166, purity: 99.80%), Cisplatin (HY-17394, purity: 99.30%) and dimethyl sulfoxide (DMSO; HY-Y0320C, purity: 99.98%) were purchased from MedChemExpress. DMSO was used as the vehicle, and the final concentration of DMSO in all cell culture systems was maintained <0.1% (v/v) to exclude solvent cytotoxicity.

Recombinant lentiviruses encoding 3-phosphoinositide-dependent protein kinase-1 (PDK1) (backbone: pCDH-CMV-MCS-EF1α-Puro) and empty vector control were purchased from Shanghai GenePharma Co., Ltd. Lentiviral particles were generated via the third-generation lentiviral packaging system in 293T cells (American Type Culture Collection). For transfection in 10-cm culture dishes, the mass ratio of transfer plasmid:packaging plasmid (psPAX2):envelope plasmid (pMD2.G) was 4:3:1, with a total plasmid amount of 8 µg per dish. Lentivirus-containing supernatant was collected after transfection. Target cells were infected with PDK1-overexpressing lentivirus (PDK1-OV) or empty vector lentivirus (EV) at a multiplicity of infection of 10 and incubated at 37˚C for 48 h. After transduction, the cells were cultured for 8 days before subsequent experimentation. Stable transfectants were then selected and maintained using 2 µg/ml puromycin (P9620; MilliporeSigma).

Cell viability was evaluated using the CCK-8 kit (Beyotime Biotechnology). Cells plated in 96-well plates were treated with various concentrations of gramine (12.5, 25, 50, 100 or 200 µM) or/and cisplatin (5, 10, 20, 40 or 80 µM) at 37˚C for 24 h. Subsequently, the culture medium was substituted with 10% CCK-8 solution prepared in 10% FBS-supplemented medium, and the plates were incubated at 37˚C for 3 h. The optical density was then measured at 450 nm using a microplate reader (Thermo Fisher Scientific, Inc.). Each group contained 6 replicate wells, and the assay was repeated 3 times biologically.

Cells (5x10²) plated in 12-well plates were allowed to grow for 5 days, followed by treatment with gramine for another 3 days. Thereafter, the colonies were fixed and stained with 0.1% crystal violet (Beijing Solarbio Science & Technology Co., Ltd.) dissolved in methanol at room temperature for 30 min. Images were captured under a light microscope. Colonies containing >50 cells were quantified using ImageJ software version 1.8.0 (National Institutes of Health). A total of 3 biological replicates were performed, and each group had 3 parallel plates.

For the EdU assay, cells grown on 12-well glass slides were treated with gramine at 37˚C for 24 h, followed by incubation with 10 µM EdU at 37˚C for 3 h. Cells were then fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100 at room temperature, and stained using the Click Reaction Mixture (Beyotime Biotechnology) according to the manufacturer's protocol. For the TUNEL assay, cells were fixed and permeabilized as aforementioned and subsequently incubated with the TUNEL reaction mixture at 37˚C (Beyotime Biotechnology). Nuclei were dyed with DAPI (Beijing Solarbio Science & Technology Co., Ltd.). Fluorescent images were visualized using a fluorescence microscope (Zeiss GmbH). The percentage of EdU-positive cells was quantified using ImageJ software. For each sample, at least 3 randomly selected microscopic fields were analyzed, with 3 biological replicates for each group.

Confluent cell monolayers were scratched using a 200 µl pipette tip to create a uniform wound gap. Cells were then cultured in serum-free medium and treated with gramine for 24 h. Wound images were imaged at 0 and 24 h using an inverted microscope (Nikon Corporation). The wound areas were measured using ImageJ software, and the wound closure rate was calculated by the formula: Wound closure rate (%)=(Area _{0 h}-Area _{24 h})/Area _{0 h} x100. For each sample, at least 3 randomly selected microscopic fields were analyzed, with 3 biological replicates for each group.

Transwell inserts (8-µm pore size, Corning, Inc.) were coated with 0.2% Matrigel (Corning, Inc.) and incubated at 37˚C for 2 h to allow gel solidification. Cells (5x10⁴) suspended in 200 µl of serum-free medium were introduced to the upper chamber, and 600 µl of complete medium containing 10% FBS was placed in the lower chamber as a chemoattractant. After overnight incubation to allow cell attachment, gramine was added to the upper chamber and the cells were incubated at 37˚C for 24 h. Non-invading cells on the upper surface of the membrane were carefully erased using cotton swabs. Cells that had invaded through the membrane and adhered to the lower surface were fixed with 4% paraformaldehyde at room temperature for 30 min, followed by staining with 0.1% crystal violet (Beyotime Biotechnology) at room temperature for 15 min. Images were acquired using a light microscope (Zeiss GmbH), and the numbers of invaded cells were quantified using ImageJ software. For each sample, at least 3 randomly selected microscopic fields were analyzed, with 3 biological replicates for each group.

Cells treated with gramine for 24 h were lysed in RIPA lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.) supplemented with PMSF (Beijing Solarbio Science & Technology Co., Ltd.) and phosphatase inhibitors (Bimake). Protein quantification was performed via the BCA assay, and identical quantities of protein (20 µg) were loaded into each lane for western blot analysis. Protein samples were then separated by 10% SDS-PAGE and transferred onto PVDF membranes (MilliporeSigma). After blocking with 5% non-fat milk for 1 h at room temperature, the membranes were incubated with primary antibodies at 4˚C overnight, followed by incubation with anti-rabbit (1:5,000; SA00001-2; Proteintech Group, Inc.) or anti-mouse (1:5,000; SA00001-1; Proteintech Group, Inc.) secondary antibodies for 1 h at room temperature. The primary antibodies used were as follows: Ki67 (1:2,000; 28074-1-AP; Proteintech Group, Inc.), Bax (1:20,000; 50599-2-Ig; Proteintech Group, Inc.), Bcl-2 (1:5,000; 68103-1-Ig; Proteintech Group, Inc.), PARP1 (1:1,000; 13371-1-AP; Proteintech Group, Inc.), cleaved PAPR1 (1:5,000; 60555-1-Ig; Proteintech Group, Inc.), N-cadherin (1:20,000; 22018-1-AP; Proteintech Group, Inc.), E-cadherin (1:20,000; 20874-1-AP; Proteintech Group, Inc.), vimentin (1:20,000; 10366-1-AP; Proteintech Group, Inc.), GAPDH (1:50,000; 60004-1-Ig; Proteintech Group, Inc.), PDK1 (1:1,000; A0834; Abclonal Biotech Co., Ltd.), AKT (1:1,000; 4691; Cell Signaling Technology, Inc.) and p-AKT (Thr308) (1:1,000; 9275; Cell Signaling Technology, Inc.). Protein bands were visualized using an enhanced chemiluminescence detection system (Thermo Fisher Scientific, Inc.) with Amersham Imager 680 (GE Healthcare) in auto-exposure mode. Band densities were quantified with ImageJ: p-AKT (Thr308) signals were normalized to total AKT, and all other protein bands were normalized to GAPDH. A total of 3 independent biological replicates were performed.

Data are presented as the mean ± standard deviation. Statistical analyses were performed using GraphPad Prism software version 8.3.1 (Dotmatics). Normality of the data were first assessed using the Shapiro-Wilk test. Comparisons between two groups were conducted using two-tailed Student's t-test, while comparisons among multiple groups were tested by one-way analysis of variance followed by Tukey's post hoc test. P<0.05 was considered to indicate statistically significant difference.

Fig. 1A depicts the chemical structure of gramine. The biological effects of gramine were evaluated in two OC cell lines (SK-OV-3 and OV-90) and normal ovarian epithelial IOSE80 cells. As shown in Fig. 1B, gramine triggered a dose-dependent suppression of cell viability across all three cell lines, with half-maximal inhibitory concentration (IC₅₀) values of 78.63 µM (SK-OV-3), 117.2 µM (OV-90) and 393.6 µM (IOSE80). The markedly lower IC₅₀ of gramine in OC cells compared with IOSE80 cells suggest a degree of selective cytotoxicity against malignant ovarian cells. Consistent with the viability data, gramine dose-dependently diminished the colony-forming capacity in OC cells (Fig. 1C). Based on these results and its minimal cytotoxicity toward normal ovarian epithelial cells, concentrations of 50 and 100 µM gramine were selected for the subsequent investigations. Additionally, the proportion of EdU-positive cells (Fig. 1D) and the expression level of Ki67 protein (Fig. 1E) were notably decreased following gramine administration. Next, it was explored whether gramine induced apoptotic cell death in OC cells. As shown in Fig. 2A, an increased number of TUNEL-positive cells was observed in gramine-treated cells. Concurrently, elevated ratios of BAX/Bcl-2 and cleaved PARP1/PAPR1 were detected after gramine exposure (Fig. 2B). These data suggest that gramine inhibits OC cell proliferation and promotes apoptosis.

Then it was assessed whether gramine affects the metastatic potential of OC cells. As shown in Fig. 3A and B, wound healing and Transwell invasion assays revealed that gramine led to reduced migration and invasion capacities, respectively. Western blot analysis further demonstrated that gramine downregulated the expressions of mesenchymal markers N-cadherin and vimentin, while concomitantly upregulating the epithelial biomarker E-cadherin (Fig. 3C). These findings suggest that gramine attenuates the invasive capacity of OC cells.

Given the vital role of PDK1 in the tumorigenesis of OC, the present study sought to investigate whether gramine impedes ovarian cancer cell growth by suppressing PDK1. Western blot analysis revealed that gramine exposure for 24 h markedly reduced PDK1 protein levels in OC cells in a concentration-dependent manner (Fig. 4A). To further clarify the functional role of PDK1 in gramine-mediated cellular responses, lentiviral transfection was performed to generate stable PDK1-overexpressing OC cell lines for subsequent rescue experiments (Fig. 4B). Functional assays validated that PDK1 overexpression effectively counteracted the suppressive effects of gramine on OC cell proliferation. Specifically, following gramine treatment, cells with increased PDK1 expression exhibited higher cellular viability (Fig. 4C), enhanced colony-forming capacity (Fig. 4D) and elevated Ki67 levels (Fig. 4E) compared with control cells. Furthermore, PDK1 overexpression partially mitigated the pro-apoptotic property of gramine. Although the proportion of TUNEL-positive cells did not reach statistical significance, ectopic PDK1 expression tended to abrogate gramine-induced apoptosis (Fig. 5A). Consistently, the increased Bax/Bcl-2 ratio induced by gramine was also rescued upon PDK1 overexpression (Fig. 5B). These data collectively suggest that gramine-mediated suppression of OC cell growth is associated with PDK1 downregulation.

Ectopic overexpression of PDK1 potentiated the migratory and invasive capacities of OC cells (Fig. 6A and B). Consistent with this phenotypic change, PDK1 overexpression also upregulated N-cadherin while downregulating E-cadherin protein levels (Fig. 6C). Notably, the inhibitory effects of gramine on cell migration and invasion were substantially abrogated following PDK1 overexpression (Fig. 6A and B). Similarly, PDK1 enrichment counteracted gramine-mediated N-cadherin reduction and E-cadherin elevation (Fig. 6C). In addition, PDK1 overexpression also effectively restored the level of p-AKT (Thr308), which was suppressed by gramine treatment (Fig. 6C). Together, these data suggest that PDK1/AKT cascade contributes to the anti-tumor activity of gramine in OC cells.

It was explored whether gramine could alleviate cisplatin resistance in OC cells. As depicted in Fig. 7A, SK-OV-3-R cells exhibited significant tolerance to cisplatin compared with parental SK-OV-3 cells, even at the maximum tested concentration of 80 µM, confirming successful establishment of the cisplatin-resistant cell model. Functional assays revealed that combined treatment with gramine (50 µM) and cisplatin significantly enhanced the sensitivity of SK-OV-3-R cells to cisplatin, as evidenced by reduced cell viability and colony-forming potential (Fig. 7B and C). Moreover, the co-treatment group presented an increased proportion of TUNEL-positive cells and impaired migration and invasion capacities (Fig. 7D and E). Mechanistically, western blot results demonstrated that combined application of gramine and cisplatin induced a more pronounced reduction in PDK1 and p-AKT (Thr308) proteins abundance compared with the cisplatin monotherapy (Fig. 7F). These data suggest that gramine may increase cisplatin responsiveness in SK-OV-3-R cells, an effect associated with reduced PDK1/AKT signaling.