Sparstolonin B exerts beneficial effects on prostate cancer by acting on the reactive oxygen species‐mediated PI3K/AKT pathway

Abstract Prostate cancer is a major health concern in males worldwide, owing to its high incidence. Sparstolonin B (SsnB), a component of the Chinese herbal medicine Sparganium stoloniferum, is used to treat many diseases. However, the effects and mechanisms of action of SsnB in prostate cancer have not yet been reported. In this study, we evaluated the effects of SsnB on cellular processes and tumour growth. In particular, we verified that SsnB could inhibit the proliferation, migration and invasion of prostate cancer cells and induce apoptosis by activating G2/M phase arrest in vitro based on a series of cytological experiments. In vivo, we found that SsnB could inhibit tumour growth in nude mouse xenograft models. We further confirmed that SsnB could repress the PI3K/AKT pathway by increasing reactive oxygen species (ROS) accumulation and oxidative stress. Collectively, SsnB inhibits tumour growth and induces apoptosis in prostate cancer via the suppression of the ROS‐mediated PI3K/AKT pathway and may be a new alternative to adjuvant therapy for prostate cancer.

Isocoumarins have anti-tumour, anticoagulant, anti-diabetes and antimicrobial bioactivities. 15,16 The special structure of SsnB makes it promising for the treatment of many diseases, especially inflammatory diseases, neurological diseases and tumours. 9 The relationship between SsnB and the progression of PCa has thus far not been explored, and the mechanism underlying its anti-tumour effect is still unclear. In this study, we evaluated the functions of SsnB in PCa with respect to proliferation, migration, F I G U R E 1 Sparstolonin B (SsnB) inhibits the proliferation, migration and invasion of prostate cancer (PCa) cells in vitro. A, Chemical structure of SsnB. B, Inhibition rates for various concentrations of SsnB on DU145 and PC-3 cells over 48 h. C, D Cell viabilities of DU145 and PC-3 after 24, 48 and 72 h of treatment. E, I Colony formation abilities of DU145 and PC-3, as detected by the colony formation assay. F, J Invasion abilities of DU145 and PC-3, as detected by the Transwell assay (scale bar: 50 μm). G, H, K Migration abilities of DU145 and PC-3, as detected by the wound healing assay (scale bar: 100 μm). *P < .05, **P < .01, ***P < .001, ****P < .0001 vs the control group; # P <.05, ## P <.01 vs groups treated with the same concentration at 24 h invasion, apoptosis, cell cycle progression and oxidative stress, both in vitro and in vivo, for the first time, providing a basis for the development of novel comprehensive therapies for PCa.

| Cell culture
The  Figure 1A) was prepared in dimethyl sulphoxide (DMSO) (Solarbio). Cells were exposed to SsnB in fresh medium at different doses, and an equal volume of DMSO solution was used for the vehicle control group.

| Cell Counting Kit-8 assay
A Cell Counting Kit-8 (CCK-8) assay (APExBIO) was used to evaluate the effects of different concentrations of SsnB on proliferation in PCa cell lines. The cells were seeded into 96-well plates at a density of 3 × 10 3 cells per well for 24 hours, and then SsnB at different concentrations from 0 µmol/L to 100 µmol/L was replenished in each well for further culture. Next, the medium in each well was replaced with 10% (V/V) CCK-8 reagent, and the plates were placed in the dark for 2 hours at 37°C. Subsequently, the optical density (OD) value for each well at 450 nm was measured using the Multi-Mode Microplate Reader (BioTek SynergyHT). Experiments were repeated independently at least three times. The IC 50 value was calculated as the concentration of SsnB when the cell inhibition rate was 50%.
Inhibition rate and cell viability were calculated using the following formulae:

| Colony formation assay
PCa cells in logarithmic growth stage were digested, used to prepare single-cell suspensions and then seeded on 60 mm cell dishes at a density of 500 cells per dish. The cells were maintained in medium with different concentrations of SsnB or DMSO until each colony contained 50 cells and was visible to the naked eye for 10 days. After washing the cells with phosphate buffered saline (PBS) (HyClone), the cells on the dish surface were fixed with 4% (V/V) paraformaldehyde (Sinopharm) for 20 minutes and stained with 1% (V/V) crystal violet reagent (Solarbio) for 15 minutes. The cells were rinsed with running water and air-dried naturally, and colonies were surveyed using an optical microscope (Olympus).

| Wound healing assay
The effects of SsnB on the migration ability of DU145 and PC-3 cells were evaluated by wound healing assays. When the confluence of PCa cells maintained in 6-well plates reached 80-90%, three wound lines were made using sterilized 200 µL pipette tips in each well.
After the cells were rinsed with PBS, SsnB solutions of different concentrations were added to the FBS-free medium for further culture.
Images of the wound area at 48 hours were collected using a phasecontrast microscope (Nikon).

| Transwell invasion assay
Matrigel (Corning, Inc) was diluted on ice with FBS-free medium at a ratio of 1:8. Each 24-well invasion chamber with 8 µm pores (Corning) was covered with 100 µL of Matrigel and incubated overnight in a 37°C incubator. After DU145 and PC-3 cells were starved with FBS-free medium for 12 hours, they were used to prepare a suspension, which was adjusted to a density of 2.5 × 10 5 cells/ml. Images of invasive PCa cells were obtained by a phase-contrast microscope.

| Detection of reactive oxygen species generation
Reactive oxygen species (ROS) levels were detected using the

| Detection of malondialdehyde, glutathione and superoxide dismutase
The Lipid Peroxidation MDA Assay Kit (Beyotime), the Micro Reduced GSH Assay Kit (Solarbio) and the Total SOD Colorimetric Assay Kit (Elabscience) were used to analyse levels of malondialdehyde (MDA), glutathione (GSH) and superoxide dismutase (SOD), respectively. All steps were performed in strict accordance with the protocols provided by the manufacturers. All levels were detected using the Multi-Mode Microplate Reader and normalized to the protein contents.

| Immunohistochemistry
The xenograft samples were fixed in 4% (V/V) paraformaldehyde, embedded in paraffin and cut into 3 μm sections.

| Statistical analyses
Image processing was performed using ImageJ, and the experimental data were evaluated by the t test, one-way analysis of variance (ANOVA) and two-way ANOVA using SPSS version 22.0 (SPSS) or GraphPad Prism version 8.3.0 (San Diego). All data are presented as means ± standard deviation. P < .05 was considered statistically significant.  Figure 1C, D). In complement with this, a colony formation assay further showed that the number of clones in the SsnB group was significantly lower than that in the control group (P < .05) ( Figure 1E, I), which was consistent with the results of the CCK-8 assay.

| SsnB reduced the proliferation, migration and invasion of PCa cells in vitro
A wound healing assay was conducted to verify the effect of SsnB on the migration of DU145 and PC-3 cells. The migration rate of DU145 in the control group, low-dose group, medium-dose group and high-dose group was 78.47% ± 6.29%, 62.89% ± 4.57%, 58.88% ± 4.01% and 40.94% ± 0.72%, respectively, and that of PC-3 in the control group, low-dose group, medium-dose group and highdose group was 62.02% ± 9.22%, 46.26% ± 4.27%, 42.66% ± 2.18% and 28.36% ± 1.28%, respectively, suggesting that the migration rate of each SsnB group was significantly decreased compared with that in the control group (P < .01 in DU145, P < .05 in PC-3). Furthermore, the inhibitory effect on migration increased as the SsnB concentration increased ( Figure 1G, H, K).
Similarly, cell invasion was significantly lower in the mediumdose and high-dose groups than in the control group, as determined by the Transwell assay (P < .01 in DU145, P < .05 in PC-3). The results also indicate that SsnB can inhibit the invasion of PCa cells in a dose-dependent manner ( Figure 1F, J).

| SsnB-induced cell apoptosis and G2/M phase arrest in vitro
As determined by Annexin V-FITC/PI staining for 10 minutes, rates of apoptosis in PC cells treated by medium-dose and high-dose SsnB for 48 hours were significantly higher than that in the control group (P < .01 in DU145, P < .001 in PC-3) (Figure 2A These findings collectively indicate that SsnB can significantly induce apoptosis in DU145 and PC-3 cells.
As is commonly known, cell cycling is closely related to apoptosis, and the stagnation of cell cycle progression often leads to apoptosis. 17 To determine the cause of apoptosis in PCa cells treated with 12.

| SsnB altered oxidative stress homeostasis in vitro
Oxidative stress contributes to the initiation and development of tumours. 18,19 Previous studies have reported that SsnB can stimulate ROS production in neuroblastoma cells. 20 We evaluated the effect In addition, the levels of MDA, GSH and SOD, related to oxidative stress, were measured. The levels of MDA (P < .05) were higher, while the levels of GSH (P < .01 in DU145, P < .05 in PC-3) and SOD (P < .05) were significantly lower, in the SsnB groups than in the control group (Figure 3D-I). These results suggest that SsnB can aggravate oxidative stress in PCa cells.

| SsnB suppressed the PI3K/AKT pathway
To explore the mechanisms underlying the biological effects of different doses of SsnB treatment for 48 hours in PCa cells, Western blotting was used to measure the protein levels of PI3K, AKT and p-AKT. The ratios of p-AKT to AKT in SsnB groups were significantly lower than those in the control group (P < .001), while there were no significant differences in the levels of PI3K and AKT between groups (P > .05) ( Figure 3J-Q). These results suggest that the functions of SsnB are mediated by the suppression of the PI3K/AKT pathway in vitro.

| The positive effects of SsnB on PCa cells could be partially reversed by the ROS scavenger Nacetylcysteine (NAC)
To further investigate the relationship between oxidative stress and the effects of SsnB on PCa-related cellular processes, the ROS *P < .05, **P < .01, ***P < .001, ****P < .0001 vs the SsnB (50 μmol/L) group scavenger NAC was used for a series of functional and pathway rescue experiments. The group treated with 50 μmol/L of SsnB and 5 mmol/L of NAC was regarded as the NAC treatment group. By means of CCK-8 assay, wound healing assay, Transwell assay and Annexin V-FITC/PI apoptotic assay, cell viability was higher (P < .001 in DU145, P < .05 in PC-3) ( Figure 4A, B), the wound area was larger (P < .01 in DU145, P < .05 in PC-3) ( Figure 4C-E), cell invasion was greater (P < .01 in DU145, P < .05 in PC-3) ( Figure 4F-H), and the apoptosis rate was lower (P < .01) ( Figure 5A-C) in the NAC group than those in the SsnB group. These results suggest that the effects of SsnB on proliferation, migration, invasion and apoptosis in DU145 and PC-3 cells can be partially reversed by NAC.
Similarly, Western blotting indicated that the ratios of p-AKT to AKT in the NAC group were significantly higher than those in the F I G U R E 5 Effects of sparstolonin B (SsnB) on apoptosis and the PI3K/AKT pathway in prostate cancer (PCa) cells could be partially reversed by the ROS scavenger N-acetylcysteine (NAC). A-C Apoptosis rates of DU145 and PC-3 in different groups. D-K Protein expression levels of PI3K, AKT and p-AKT in different groups. *P < .05, **P < .01, ***P < .001, ****P < .0001 vs the SsnB (50 μmol/L) group SsnB group (P < .01), while the levels of PI3K and AKT did not differ significantly between groups (P > .05) ( Figure 5D-K). These results reveal that the inhibitory effect of SsnB on the PI3K/AKT pathway in PCa cells can be partially reversed by NAC. Taken together, SsnB may inhibit cellular processes in PCa by suppressing the ROSmediated PI3K/AKT pathway.

| SsnB impeded the xenograft tumour growth in vivo
To determine the role of SsnB in the growth of PCa in vivo, xenograft models were established using PC-3 cells (Figure 6A, B). Although there was no statistically significant difference in the average body F I G U R E 6 Sparstolonin B (SsnB) impeded xenograft tumour growth in vivo. A, B Xenograft tumours in nude mouse models. C, Body weights of nude mice. D, E Volumes and wet weights of xenograft tumours in different groups. F-H, Expression levels of Ki67 and proliferating cell nuclear antigen (PCNA) in xenograft tumours detected by an immunohistochemical assay (scale bar: 50 μm). *P < .05, ***P < .001 vs the control group weight of nude mice between different groups (P > .05) (Figure 6C), the volumes of xenograft tumours in the SsnB group were significantly smaller than those in the control group (P < .05) ( Figure 6D).
Additionally, the wet weights of xenograft tumours in the SsnB group were significantly lighter than those in the control group (P < .001) ( Figure 6E). Moreover, the expression levels of Ki67 and PCNA in the SsnB group were significantly lower than those in the control group in the immunohistochemistry assay (P < .001) ( Figure 6F-H).
These data indicate that SsnB plays a crucial role in impeding tumour growth in vivo.

| D ISCUSS I ON
The high incidence of PCa is an important issue worldwide. Although in melanoma. 30 On the one hand, the essence of SsnB is a polyphenol with two core components of xanthone and isocoumarin. 10 Polyphenols are well known for their regulation effects on ROS production. [31][32][33][34] In line with this, it has been found that SsnB can promote the production of ROS in neuroblastoma, 20 indicating that SsnB may be a kind of polyphenols with pro-oxidant effect, which may increase the production of hydroxyl radicals through protonophoric effect and self-oxidation to form quinones, increase ROS generation, reduce mitochondrial membrane potential and induce cell apoptosis. 35 On the other hand, SsnB has been widely mentioned as one of toll-like receptor (TLR) inhibitors, especially TLR-2 and TLR-4 in many literatures. 36,37 TLRs are believed to play a crucial role in tumourigenesis and progression, 38  We subsequently evaluated the mechanisms by which SsnB protects against PCa. We focused on oxidative stress, a key biological process involved in the occurrence and development of many disorders, such as cardiovascular diseases, 44 neurological diseases, 45 psychiatric disorders, 46 reproductive diseases, 47 allergic diseases 48 and tumours. 49 ROS can lead to DNA mutations associated with tumourigenesis, and the levels of ROS are usually elevated in tumour cells; generally, the antioxidant system of tumour cells can also be activated, to maintain stability of the redox system. 50 It is believed that this plays a positive role in tumour treatment, 51  The PI3K/AKT pathway is closely related to tumour-related processes 53 and tumour resistance. 54 As a key pathway in PCa, it exhibits complex interactive crosstalk with other oncogenic signalling pathways. 55 The PI3K/AKT pathway is activated by class 1A PI3Ks, and the phosphorylation of AKT results in the regulation of cellular processes. 56 It is clear that the PI3K/AKT pathway affects cell proliferation, migration, invasion and apoptosis in PCa, [57][58][59][60] suggesting that the inhibition of this pathway could be a therapeutic strategy for PCa.
Our experimental results showed that SsnB could inhibit the PI3K/ AKT pathway and increase ROS levels, and the inhibitory effect could be partially reversed by the ROS scavenger NAC. These results indicate that SsnB can suppress the ROS-mediated PI3K/AKT pathway.

CO N FLI C T O F I NTE R E S T
There are no potential conflicts of interest to declare. Yongsheng Song: Conceptualization (lead); Supervision (lead).

DATA AVA I L A B I L I T Y S TAT E M E N T
All data supporting the findings of our study are available from the corresponding author upon reasonable request.