Docetaxel was purchased from Selleck Chemicals (Cat# RP56976). RPMI 1640 medium was purchased from Gibco (Cat# 8120018). Fetal bovine serum was purchased from Gemini (Cat# 900-108). Charcoal stripped FBS was purchased from Biological Industries (Cat# 04-201-1A). Thiazolyl blue tetrazolium bromide (Cat# M8180) and crystal violet stain solution (Cat# G1063) were purchased from Solarbio life sciences. Human apoptosis array kit was from R&D Systems (Cat# ARY009). NuPAGETM 10% Bis-Tris Gel (Cat# NP0302BOX), eBioscience™ Annexin V-FITC apoptosis detection kit (Cat# BMS500FI-300), FxCycle™ PI/RNase staining solution (Cat# F10797) and Vybrant cell-labeling solutions (V22885) were purchased from Invitrogen. All antibodies used in this study were purchased from Proteintech Group (CDK4, Cat# 66950-1-lg; CDK6, Cat# 66278-1-lg; Cyclin D1, Cat# 26939-1-AP; RB1, Cat# 10048-1-lg; E2F1, Cat# 66515-1-lg; Survivin, Cat# 66495-1-lg; p21, Cat# 10355-1-AP). Eeq was isolated and identified from the marine sponge-derived fungus Fusarium equiseti SCSIO 41019 by various chromatographic and comprehensive spectroscopic methods in our previous study (Chen et al., 2019), respectively. It was determined to have ≥95% purity by analytical HPLC.

LNCaP, 22Rv1, DU145, PC-3, and WPMY-1 cells were obtained from the National Collection of Authenticated Cell Cultures (Shanghai, China). All cell lines were identified by STR, and the cells used in the experiment were within 15 passages. LNCaP and 22Rv1 cells were maintained in RPMI 1640 supplemented with 10% (v/v) fetal bovine serum (FBS), 100 units/mL penicillin, and 100 μg/μL streptomycin; DU145 cells were cultured in MEM plus with the same additives; PC-3 cells were cultured in DMEM F12 plus with the same additives; WPMY-1 cell line was maintained in DMEM (Gibco, China) supplemented with 5% (v/v) fetal bovine serum (FBS) (Biological Industries, Israel), 100 units/mL penicillin, and 100 μg/μL streptomycin.

Cell viability was analyzed using MTT assay as previously described (Wang et al., 2021). In brief, cells were seeded overnight in a 96-well plate at a density of 5*10³ per well and then treated with a range of concentrations of Eeq for the intended time. After the cells were exposed to Eeq for a certain time, the MTT solution was added to the cell culture wells for 4 h. Then, OD₅₇₀ values were detected by using a Hybrid Multi-Mode Reader (Synergy H1, BioTek). The experiment was repeated three times independently. IC₅₀ was calculated by using GraphPad 9.0 software. The degree of selective cytotoxicity index was expressed as SI = IC₅₀ in normal cells/IC₅₀ in tumor cells.

PC-3 cells were seeded in a 6-well plate at a density of 1000 per well overnight, and then they were treated with DMSO (0.1%, v/v) and docetaxel (1 μM), Eeq (2.5, 5, 10 μM) for demanded time, respectively. The culture medium was changed every 72 h until the obvious cell cloning colonies were formed about 15 days later, and then the cells were fixed with 4% formaldehyde for 30 min, discarded 4% formaldehyde and washed cells with PBS buffer, and stained the colonies with crystal violet stain solution. After the cells had been stained for 30 min, we discarded the staining solution, washed the cells with PBS buffer, and then took colonies’ photos with a colony counter (GelCount, Oxford Optronix). The experiment was repeated three times independently.

PC-3 cells were seeded in a 6-well plate at a density of 2.0*10⁵ per well overnight and treated with DMSO (0.1%, v/v), docetaxel (1 μM), Eeq (2.5, 5, and 10 μM), respectively, for 48 h. The cells were collected and stained according to the instructions of eBioscience™ Annexin V-FITC apoptosis detection kit and FxCycle™ PI/RNase staining solution, respectively. Then, cell apoptosis and cell cycle distribution were detected using flow cytometry (NovoCyte, Agilent). This experiment was repeated three times independently.

A gradient concentration of Eeq (1.25, 2.5, 5, 10, 20, and 40 μM) solution was prepared with 10% FBS DMEM F12 medium in a 96 well U-shaped plate, and then these solutions were transferred to the lower chamber in CIM-plate 16 (5665817001, Agilent) with 2 wells for each concentration. Next, the upper chamber of the CIM-plate 16 was mounted directly above the lower chamber to form a complete plate. PC-3 cells were seeded in the upper chamber of the CIM-plate 16 at a density of 2.5*10⁵. Eeq was then added to the upper chamber so that the concentration of Eeq matched that of the lower chamber. After that, the CIM-plate 16 was installed on a real-time cell analyzer (xCELLigence RTCA DP Instrument, Agilent) to detect the effect of Eeq on the migration of PC-3 cells.

PC-3 cells were seeded in T75 Flask at a density of 5.0*10⁶ for 48 h and treated with DMSO (0.1%, v/v) and Eeq (10 μM) for 48 h, respectively. Then, cells were collected, and proteins were extracted and analyzed according to the instructions of the human apoptosis array kit.

qPCR was performed as described previously (Wang et al., 2021). In brief, PC3 cells were seeded into 6-well plates and grown in DMEM F12 medium containing 10% FBS. After attachment, cells were treated with Eeq for 24 h. Then RNA was extracted and purified using the TRIzol reagent (15596018, Life technologies). cDNA was prepared from 1 μg of RNA with RevertAid Master Mix (M1632, Thermo Fisher Scientific). Diluted cDNA was used to perform quantitative RT-PCR (LightCycler96, Roche) using PowerUp SYBR Green master mix (A25742, applied biosystems) with GAPDH as the internal standard. Primers for quantitative RT-PCR were listed in Table 1. Experiments were performed in triplicate.

PC-3 cells were seeded in 60 mm dishes at a density of 1.0*10⁶ for 48 h. Then, cells received a fresh medium containing the indicated compounds, docetaxel (1 μM), and the indicated concentrations of Eeq (2.5, 5, and 10 μM). The whole cell extracts were prepared after 48 h by using the mixture of RIPA (R0020, Solarbio life science) and PMSF (P0100, Solarbio life science). Proteins were analyzed on 10% SDS-PAGE gel sand transferred to nitrocellulose membranes. In this study, 30 μg of total protein was used to detect all proteins except DR5, which was measured by using 60–80 μg of total protein. All information of antibodies is listed in the section on reagents and antibodies. Images were captured using the Chemidoc CD Touch (Bio-Rad, United States), and images were analyzed and processed using the Image Lab 6.0 (Bio-Rad, Chinese edition).

PC-3 cells were seeded in a 6-well plate at a density of 2.0*10⁵ for 48 h, and then cells were transfected with si-RNA (small interfering RNA sequences are shown in Table 2) by using Lipofectamine 2000 (11668019, Thermo Fisher Scientific) according to the manufacturer’s manual. After 48 h of transfection, Eeq was added to the 6-well plate, and the cells were exposed to the compound for another 48 h. Then, apoptosis was detected using flow cytometry.

Animal experiments were performed according to procedures approved by the Guangxi University of Chinese Medicine Institutional Animal Ethical and Welfare Committee (Approval code: 20210603-071). Male BALB/c nude mice (Hunan SJA laboratory animal Co., Ltd., China) of 5–6 weeks old were acclimated to a sterilized normal diet and water for seven days. Mice were inoculated subcutaneously with 5*10⁵ androgen-independent PC-3 human prostate cancer cells. The PC-3 cell line was purchased from Cell Bank/Stem Cell Bank, The Committee of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China), where the cells were authenticated by STR DNA profiling and tested as mycoplasma free. The intervention started when tumors reached a volume of 50–100 mm³ about one week after inoculation. Mice were randomly assigned into three groups (n = 6-7): vehicle, 10 mg/kg docetaxel, 20 mg/kg, and 40 mg/kg Eeq. Docetaxel and Eeq were dissolved in a solvent containing 5%DMSO, 30% PEG300, 5% Tween 80 and dd H₂O. Docetaxel was intraperitoneally administered twice a week for four weeks, and Eeq was administered once a day throughout the study. Tumor size was measured twice a week with calipers. Tumor volume was calculated by using the formula: π/6*length*width*width (Wang et al., 2016). Mouse body weight was measured twice a week. Mice were sacrificed when they had received 4-week intervention treatment in observance of the institutional guideline on tumor size. Xenograft tumors were harvested, weighed, and fixed with 4% paraformaldehyde and/or snap frozen and stored in liquid nitrogen.

All analyses were conducted using GraphPad Prism 9 (GraphPad Software, United States). All results were presented as mean ± standard deviation (SD) and analyzed using one-way ANOVA. Comparisons between the groups were conducted using Dunnett’s t-test. Level for statistical significance was set at *p < 0.05, **p < 0.01.

Eeq inhibited the cell viability of prostate cancer cells (LNCaP, 22Rv1, DU145, and PC-3) and normal prostatic cells (WPMY-1) in varying degrees in a dose-dependent manner by MTT assay, among which, it showed the most significant effect on PC-3 cells (IC₅₀ = 4.43 ± 0.24 μM). The selective cytotoxicity index (SI) of Eeq for PC-3, DU145, 22Rv1 and LNCaP cells were 4.55, 1.64, 1.38, and 0.57, respectively, suggesting that Eeq has the best selectivity for PC-3 cells (Figure 1B). Then, we used the MTT assay to further investigate the effect of Eeq on the viability of PC-3 cells. We found that the cytotoxicity of Eeq on PC-3 cells was time-dependent (Figure 1C). Next, we performed a plate clone formation assay to investigate the effect of Eeq on the proliferation of PC-3 cells. The results showed that it could significantly inhibit the clone formation of PC-3 cells in a dose-dependent manner (Figures 1D and E). Notably, the PC-3 cell line is a prostate cancer cell that does not express the androgen receptor, and these findings suggest that Eeq does not exert its anti-prostate cancer effects through modulation of the androgen receptor signaling pathway. It must then exert its effects through other pathways.

In order to reveal how it inhibited the proliferation of prostate cancer cells, we detected the cell cycle distribution of PC-3 cells. As shown in Figures 1F and G, when being treated with docetaxel, which is an M-phase blocker, a large number of PC-3 cells were arrested at the M phase, and the proportion of G2/M phase cells increased sharply. However, unlike in the case of docetaxel, the percentage of the G1 phase increased significantly in the PC-3 cells which were treated with Eeq. These findings point to the fact that Eeq blocks the cell cycle at the G1 phase, resulting in the inability of cells to proliferate.

Cyclin D, CDK4, and CDK6 are fundamental drivers of the cell cycle in driving G1 to S phase transition (Goel et al., 2017). In order to clarify the mechanism that Eeq blocked the cell cycle in the G1 phase, we performed Western blot to detect the expression of the proteins above. As shown in Figure 2, Eeq did reduce the expression of Cyclin D, CDK4, and CDK6 proteins while upregulating the expression of p21 and p27 proteins (Figures 2D–H), which are also key roles in regulating the cell cycle from G1 phase to S phase (Coqueret, 2003). Furthermore, PI3K/Akt/p21 signaling axis is a key pathway to regulate the cell cycle (Chang et al., 2003). In the present study, Eeq inhibited PI3K and Akt protein expression by reducing Akt phosphorylation at Ser473 (Figures 2A–C). Finally, it declined the expression of survivin protein, leading to cell death (Figure 2I). Based on the findings above, it can be concluded that Eeq inhibited the function of the PI3K/Akt/p21 signaling pathway, which, in turn, blocked the G1 phase of the cell cycle, resulting in the inability of cells to divide and the eventual loss of proliferative capacity.

On the other hand, we found that Eeq inhibited PC-3 cell migration with an IC₅₀ value of 6.80 ± 0.31 μM by real-time cell analysis (Figure 3A). In addition to directly corresponding to the cell cycle, the PI3K/Akt signaling pathway also plays a key role in cell invasion and migration (Hamidi et al., 2017). It is well known that beta-catenin is involved in WNT and/or PI3K/Akt signaling pathways which regulate cancer cell migration, thereby it is considered a target for the treatment of cancer (Tang et al., 2019; Zhang et al., 2020). We found that Eeq decreased the protein expression of β-catenin and N-cadherin and increased the expression of E-cadherin (Figures 3B–D), while it showed no impact on WNT5A/5B (Supplementary Figure S2F). These findings may provide evidence that Eeq attenuated epithelial–mesenchymal transitions through PI3K/Akt/beta-catenin/cadherin in prostate cancer cells, thereby inhibiting the migration of PC-3 cells.

To further identify how Eeq produced cytotoxicity, we used flow cytometry to detect the apoptosis of PC-3 cells. As shown in Figures 4A and B, Eeq induced cell apoptosis in a dose-dependent manner with docetaxel acting as a positive control. In order to clarify how Eeq induced apoptosis, we performed a human apoptosis array. As shown in Figures 4C and D, Bcl-x, cleaved caspase-3, DR5, p21, and p27, were upregulated after Eeq had been treated for 48 h. However, the survivin protein that promotes cell survival was downregulated. Among the apoptosis proteins encapsulated in the human apoptosis array, DR4, DR5, TNFRSF6, and TNFRSF1A are proteins involved in the TNF signal transduction pathway, which is an important pathway that mediates apoptosis. DR5 was upregulated after Eeq had been treated for 48 h, while DR4, TNFRSF6, and TNFRSF1A were not upregulated by Eeq (Figure 4C). Therefore, we studied DR5 in the follow-up work. We noticed that DR5 was a cell membrane receptor that mediated cell apoptosis. These findings inspired us to speculate that Eeq mediated apoptosis by driving the DR5 signaling pathway. Then, in order to confirm this hypothesis, we conducted a series of experiments.

Activation of caspase-8 and caspase-3 is the key link in the induction of apoptosis by DR5 (Muñoz-Pinedo and López-Rivas, 2018). In this study, caspase-8 and caspase-3 were activated, as evidenced by a decrease in the full length of caspase-8/3 and an increase in cleaved caspase-8 (Figures 5A and B). Furthermore, we found Eeq increased expression of DR5 on both mRNA and protein levels (Figures 5C and D). Most importantly, the apoptotic cells induced by Eeq were significantly reduced (Figure 5H) when DR5 expression was silenced by small interfering RNA (Figures 5E–G). Taken together, Eeq induced PC-3 cell apoptosis through the DR5 signaling pathway.

In order to further evaluate the anti-PCa effect of Eeq in vivo, we constructed a PCa xenograft model by subcutaneously injecting PC-3 cells into the right flank of the mice. In the first experiment, we divided the model mice into vehicle group, docetaxel group (10 mg/kg), and Eeq group (10–20 mg/kg). The Eeq concentrations tested in mice refer to one of our previous studies. Ilicicolin A is also a marine compound which was isolated from the Beibu Gulf coral–derived fungus Acremonium sclerotigenum GXIMD 02501. It showed antiproliferative activity in human prostate cancer at 2.5–10 μM in vitro and at 10 mg/kg in vivo (Guo et al., 2021). The inhibitory activity of Eeq on prostate cancer proliferation in vitro is similar to that of ilicicolin A, so we set the 10 mg/kg dose as the initial dose of Eeq in vivo. We found that Eeq showed tumor growth inhibition in only half the number of mice at a dose of 10–20 mg/kg. In general, the tumor growth inhibition of 10–20 mg/kg Eeq was less effective than that of 10 mg/kg docetaxel in terms of tumor size and weight (Supplementary Figure S1). Then, we carried out the second experiment and changed the dose of Eeq to 20–40 mg/kg. Herein, 20–40 mg/kg Eeq dramatically inhibited tumor growth in mice, evidenced by a sharp decline in tumor size and weight (Figures 6A–C). Moreover, all mice showed good tolerance to 20–40 mg/kg Eeq, and no mice died during the experiment. There were no significant changes in body weight and organ index (Figure 6D; Supplementary Figures S2A–E). Furthermore, we also examined mice tumor tissues and found that Eeq also promoted DR5 and E-cadherin while inhibiting expression of caspase 8 (full length), surviving, and N-cadherin in vivo (Figures 6E–G). However, the expression of PI3K and Akt proteins in mice tumor tissue was not inhibited as in vitro (Figure 6H), suggesting that Eeq could not affect this signaling pathway in mice. Based on the findings above, we confirm that Eeq played an anti-PCa role through DR5 and beta-catenin/E-cadherin signaling pathways.