Sparstolonin B Has Anti-tumour Effects On Prostate Cancer Via The Ros-mediated PI3K/AKT Pathway

Background:Prostate cancer（PCa）is a major health concern worldwide owing to its increasing morbidity and mortality.Among tumours affecting males,the incidence rate of PCa ranks first and second in the United States and Europe,respectively,and it has the second and third highest death rates in the United States and Europe,respectively.In China,the incidence rate of PCa occupies the seventh place in male malignant tumours.Although the incidence is far lower than that of western countries,with the gradual deepening of the understanding of this disease,the incidence of PCa has also shown an upward trend in China.A wide range of risk factors for PCa have been identified,including family history,hormone levels,ethnicity,aging,oxidative stress,sexually transmitted diseases,diet,smoking,environmental agents,occupation,and sexual activity.However,the molecular biological mechanism underlying PCa occurrence and progression is not clear.Current therapeutic regimens of PCa mainly include radical therapy,chemotherapy,and endocrine therapy.The formulation of treatment plan is related to the type and status of PCa,but no matter which therapeutic regimens more or less have various side effects or problems that cannot be solved by itself.Accordingly,an effectual and safe new strategy for the treatment of PCa is urgently needed.Sparganium stoloniferum,an aquatic herb found in North and East China,is used to regulate menstruation,and to promote galactosis and spasmolysis in traditional Chinese medicine.Sparstolonin B（SsnB）has been extracted from the rhizomes of this plant.Both X-ray crystallography and nuclear magnetic resonance spectroscopy have revealed that SsnB,whose structural formula is C₁₅H₈O₄,is a polyphenol containing two core components of xanthone and isocoumarin.Xanthones have established antioxidant,anti-inflammatory,immunomodulatory,anti-tumour,antimicrobial,anticholinesterase,and anticonvulsant functions.Isocoumarins have anti-tumour,anticoagulant,anti-diabetes,and antimicrobial bioactivities.The special structure that SsnB makes it promising for the treatment of many diseases,especially inflammatory diseases,neurological diseases,and tumours.Objective:The relationship between SsnB and the progression of PCa has not been explored to date,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 the biological behaviour of proliferation,migration,invasion,and apoptosis;we explored whether SsnB worked via reactive oxygen species（ROS）-mediated phosphoinositide 3-kinase/AKT（PI3K/AKT）pathway;we observed whether SsnB also exhibited anti-tumour growth effects in vivo for the first time,providing a basis for SsnB as a novel monomer to be one of the potential candidates in the comprehensive therapies for PCa.Methods:In this study,SsnB solutions in fresh medium at different doses were used for SsnB treatment groups,and an equal volume of dimethyl sulfoxide（DMSO）solution was used for the vehicle control group.PCa cell lines DU145 and PC-3 were maintained to carry out a series of cytological experiments.The cell counting kit-8（CCK-8）assay and the colony formation assay were conducted to verify the effect of SsnB on the proliferation of DU145 and PC-3 cells.The wound healing assay and the Transwell assay was performed to detect the effect of SsnB on migration and invasion of PCa cells,respectively.Moreover,the regulation of SsnB on the cell cycle of DU145 and PC-3 cells was detected by flow cytometry,and the influence of SsnB on apopotosis was detected using the Annexin V-fluorescein isothiocyanate/propidium iodide（Annexin V-FITC/PI）apoptotic assay and the western blot assay of apoptosis-related proteins such as Bax,Bcl-2,Caspase 3 and cleaved-Caspase 3.In order to explore whether the anti-tumour function of SsnB in DU145 and PC-3cells was presented via ROS mediated-PI3K/AKT pathway,we further tested the ROS level using the fluorescent microscope and the Multi-Mode Microplate Reader,and the levels of malondialdehyde（MDA）,glutathione（GSH）,and superoxide dismutase（SOD）were measured using the Multi-Mode Microplate Reader to judge the oxidative stress state of PCa cells.The western blot assay was conducted to detect the protein levels of PI3K,AKT,and p-AKT.Then,the ROS scavenger N-acetylcysteine（NAC）was used for a series of experiments on proliferation,migration,invasion,apoptosis,and the PI3K/AKT pathway,to verify whether NAC could rescue the effects of SsnB in PCa cells.In addition,1×10⁷ PC-3 cells were subcutaneously injected into the axilla region of BALB/c nude mice,and the mice were randomly divided into a vehicle control group and an SsnB group.Weights and tumour sizes were measured regularly.The mice were sacrificed with euthanasia and xenograft tumours were collected for wet weight measurement and preparation into slices.The immunohistochemical assay was used to detect the expressions of Ki67 and proliferating cell nuclear antigen（PCNA）in xenograft tumours,and demonstrate whether SsnB could inhibit the growth of prostate cancer in vivo.Results:CCK-8 results show that the inhibitory effect of SsnB on the proliferation of DU145 and PC-3 cells increased gradually as the concentration and treatment duration increased,indicating both concentration-and time-dependent effects.Complementally,the colony formation assay further showed that the number of clones in the SsnB group was significantly lower than that in the control group,which was consistent with the results of the CCK-8 assay.In the wound healing assay,the wound areas of these two PCa cell lines were significantly smaller in the SsnB groups than in the control group.Similarly,Transwell results show that cell invasion was significantly lower in the SsnB groups than in the control group.Moreover,SsnB increased the proportions of DU145and PC-3 cells in G2/M phase,accompanied by a decrease in G0/G1 phase in DU145cells.As determined by Annexin V-FITC/PI staining,rates of apoptosis in PC cells were significantly higher in SsnB groups than in the control group.A western blot assay illustrated that SsnB treatment increased protein expression levels of Bax and cleaved-Caspase 3 in DU145 and PC-3 and decreased the level of Bcl-2,with no change the level of Caspase 3.As expected,green fluorescence intensities representing ROS levels in SsnB groups were significantly increased in DU145 and PC-3 cells.Compared to levels in the control group,levels of MDA were higher while levels of GSH and SOD were significantly lower in the SsnB groups.Levels of p-AKT in SsnB groups were significantly lower than those in the control group,while there were no significant differences in the levels of PI3K and AKT between groups.In rescue experiments,by means of CCK-8 assay,wound healing assay,Transwell assay,and Annexin V-FITC/PI apoptotic assay,cell viability was higher,the wound area was larger,cell invasion was greater,and apoptosis rate was lower in the SsnB+NAC group than those in the SsnB group.Western blotting indicated that the protein expression levels of p-AKT in the SsnB+NAC group were significantly higher than those in the SsnB group,while the levels of PI3K and AKT did not differ significantly between groups.Although there was no statistically significant difference in the average body weight of nude mice between different groups,the volumes of xenograft tumours in the SsnB group were significantly smaller than those in the control group.Additionally,the wet weights of xenograft tumours in the SsnB group were significantly lighter than those in the control group.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.Conclusion:We provide the first clear demonstration of the role and mechanism of action of SsnB in PCa.SsnB could inhibit the proliferation,migration,and invasion of PCa cells,induce apoptosis by G2/M phase arrest in vitro.Furthermore,SsnB can disrupt oxidative stress homeostasis by increasing ROS levels and thereby inhibit the PI3K/AKT pathway.The results of function and pathway experiments can be partially reversed by NAC.These indicate that the beneficial effects of SsnB on tumour growth and apoptosis in PCa are mediated by the suppression of the ROS-mediated PI3K/AKT pathway.It is worth mentioning that SsnB inhibits tumour growth in vivo.Hence,SsnB may be a new alternative adjuvant therapy for PCa.