The potential antitumor activity of polysaccharide extracted from polygonatumsibiricum on human prostate cancer PC-3 cells 

The potential antitumor activity of polysaccharide extracted from polygonatumsibiricum on human prostate cancer PC-3 cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The potential antitumor activity of polysaccharide extracted from polygonatumsibiricum on human prostate cancer PC-3 cells Guobin Zhao, Yan Zhou, Yuhong Tang, Muhammad Abbas, Shaowen Dong, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4668118/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 3 You are reading this latest preprint version Abstract Polysaccharide extracted from polygonatum sibiricum (PSP) is a traditional herbal medicine component thought to have therapeutic effects on many diseases. In our study, we initially investigated the role of PSP in the development of human prostate cancer PC-3 cells and explored the potential mechanisms. First, the different concentrations of PSP were used to act on human prostate cancer cells PC-3, It was found that the proliferation, invasiveness, and migratory ability of PC-3 cells were significantly inhibited with increasing concentrations of PSP; the cell apoptosis rate and expression of caspase-3 increased with increasing concentrations of PSP; and the cell cycle was arrested in the S phase. In addition, we found that the expression of the MDR-1 gene and its encoding protein P-gp in PC-3 cells decreased after the treatment with PSP, so PSP might reverse multidrug resistance in PC-3 cells. Second, the possible mechanism of PSP action on PC-3 cells has been investigated, and the results showed that phosphorylated P65, PI3K, and AKT decreased in a concentration-dependent manner, as P65, PI3K, and AKT are molecules of the NF-kB and PI3K/Akt signaling pathways, so the potential mechanism of PSP on prostate cancer cells might be mediated by the PI3K/Akt and NF-kB signaling pathway simultaneously. PSP PC-3 cells invasiveness migratory ability P65 PI3K and AKT Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Prostate cancer is the second-leading cause of cancer-related deaths in men worldwide 1 . For early-stage prostate cancer, laparoscopic radical prostatectomy can achieve a radical treatment effect that has the characteristics of less expensive, minimally invasive 2 and quick recovery after operation. For relapse or metastatic prostate cancer, radical surgery is not suitable, while Androgen deprivation therapy (ADT) is the standard-of-care therapy at present 3 , aiming at removing androgens and inhibiting the activity of the androgens to delay the progression of prostate cancer. However, most prostate cancer patients might gradually become insensitive or ineffective to androgen blocking, thus developing castration-resistant prostate cancer (CRPC) with high morbidity and mortality 4 . It is very difficult for clinical treatment because of the natural or acquired drug resistance of CRPC, and the prognosis is poor. Therefore, it is necessary to find a new approach to effectively treat these patients with advanced prostate cancer. Recently, many studies have shown that polygonatum sibiricum polysaccharides(PSP) have many pharmacological applications and biological activities 5 and are widely used in treatments against diabetes mellitus and its complications 6 , 7 , Hypoplipidemic 8 , anti-atherosclerosis 8 , 9 , anti-osteoporosis 10 and cancers 11 , 12 . Of these, the possible anti-tumor effect of PSP has attracted the interest of scientists because it is less toxic and has no side effects. One study indicated that PSP could significantly inhibit the growth of prostate-CAF (cancer-associated fibroblasts and may improve the efficacy of cancer therapy 13 . Another study showed that PSP could induce HepG2 cell apoptosis, and arresting the cell cycle at the G1 phase might have potential antitumor effects 14 . Several studies also suggested that PSP could hold human gastric cancer HGC-27 cells, esophageal cancer ECA-109 cells, and colorectal cancer HCT-8 cells in the S stage, promoting their apoptosis 5 , 15 . How PSP influenced the prostate cancer PC-3 cell cycle and induced apoptosis? It is necessary to further explore the anti-tumor mechanisms. Studies have indicated that phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/AKT) signaling pathway is one of the key signaling pathways involved in the regulation of cell cycle 16 . Most studies also found that PI3K/AKT signaling plays a significant role in prostate cancer tumorigenesis and therapy, which participate in the apoptosis, proliferation, metastasis, and invasion of prostate cells 17 – 21 . So, our hypothesis is that whether PSP affects the biological behavior of prostate cancer cells and whether the mechanism is related to the PI3K signaling pathway. In this study, we will explore the function of PSP on prostate cancer PC-3 cells and elucidate its possible mechanism. Materials and methods Cell culture and reagents Human prostate cancer PC-3 cells were purchased from Wuhan Procell Life Science & Technology Co., Ltd. Cells were cultured in Ham's F-12K supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 mg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO2. PSP was provided by Yuanye Biotechnology Co., Ltd. The compounds were dissolved in PBS to 20 mg/ml as a stock solution, stored at -20°C for in vitro studies, and diluted wit h medium before each assay. Cell Counting kit-8(CCK-8) assay The effect of PSP on cell growth was determined using the CCK-8 assay. Cells were seeded into 96-well tissue plates at a density of 1×10 4 cells/well and incubated with various concentrations of PSP for 24 h, 48 h, or 72 h. Untreated cells served as the control group. After treatment, 10µl of CCK-8 reagent was added to each well and then incubated for an additional 4 hours at 37°C. The cell viability was determined by measuring the absorbance at 450 nm using a microplate reader. Flow cytometry Based on the results of the CCK-8 assay, PC-3 cells were plated into 6-well plates (2×10 5 cells/well) and treated with various concentrations of PSP solution for 72 h. The apoptosis rate of PC-3 cells was evaluated using the Annexin V-FITC/PI Apoptosis Detection Kit according to the instructions from the manufacturer. The cultured cells were collected, washed with cold PBS, and resuspended in binding buffer. 5 µl of Annexin V-FITC and 5 µl of PI were added to the buffer and incubated at room temperature for 15 min in the dark. Annexin V-FITC binds to phosphatidylserine on the outer membrane of apoptotic cells, while PI enters and stains cells with compromised membrane integrity and then binds to and labels DNA. A flow cytometer (BEAMDIAG; BeamCyte-1026, China) was used for data collection, and FlowJo software for data analysis. For the cell cycle assay, PC-3 cells were digested by trypsin, washed with cold PBS, and fixed with 70% alcohol at 4°C overnight. Subsequently, the fixed cells were collected and resuspended in cold PBS, then the cells were stained with PI and incubated for 30 min at 37°C in the dark. A flow cytometer (BEAMDIAG; BeamCyte-1026, China) was used for data collection, and FlowJo software for data analysis. Transwell assay A Transwell assay was used to analyze cell invasion and/or migration. Cells treated with PSP suspended in FBS-free medium were seeded in 24-well plates coated with or without Matrigel in the upper compartment, and the lower compartment was supplemented with 10% FBS. After 24 hours of incubation, non-invaded or non-migrated cells across the membranes were carefully removed with cotton swabs, while cells across the membrane were fixed with carbinol and then stained using Giemsa. After that, cells were calculated under a microscope. Western blot Cells treated with PSP were harvested and lysed with RIPA, and protein levels were measured with a BCA quantification kit. The lysates were subjected to 10% SDS-PAGE and transferred to the PVDF membrane. Membranes were probed at 4°C in the presence of 1:1000 dilutions of primary antibodies, including Akt, p-Akt, PI3K, p-PI3K, caspase-3, P65, and p-P65. After being washed three times with PBS-T, the membranes were incubated with HRP-labelled sheep anti-mouse antibody for 1 h. Finally, the blots were visualized with enhanced chemiluminescence. Real-time RT-PCR PC-3 cells were treated with different concentrations of PSP for 72 hours, then the total RNA was extracted using a Trizol reagent, and the RNA concentration was determined using NanoDrop 2000. 1 µg of RNA was reverse transcribed into cDNA with the SuperRT cDNA Synthesis Kit. GAPDH was used as an internal control. Levels of MDR1 mRNA were assessed using the UltraSYBR Mixture (Low ROX) (Beijing Comwin Biotech Co., Ltd.). The data were normalized relative to GAPDH by using the following formula: relative mRNA expression = 2-△△Ct, where Ct is the cycle threshold. Immunohistochemistry (IHC) PSP-treated cell slides were prepared, followed by serum blocking for 30 minutes at 37°C and staining with anti-MDR1/P-gp antibodies overnight at 4°C. Then the slides were incubated with HRP-conjugated secondary anti-rabbit IgG at 37°C for 40 minutes. Slides were visualized with diaminobenzidine (DAB), counterstained with hematoxylin, and observed. Statistical analysis All experiments were conducted three times. The data were analyzed by SPSS 19.0 statistical software. The experimental results were expressed as the mean standard deviation. The difference was P < 0.05. Results PSP inhibited the proliferation of PC-3 cells The CCK-8 assay was used to investigate whether PSP inhibits the growth of PC-3 cells at the appropriate inhibitory concentration. PC-3 cells were exposed to various concentrations of PSP (0 ug/mL, 125 ug/mL, 250 ug/mL, 500 ug/mL, 1 mg/mL, 2 mg/mL, and 4 mg/mL) for 24 h, 48 h, and 72 h. As shown in Fig. 1 , At 24 h, no obvious inhibition was observed at different concentrations. At 48 h, the inhibition increased with the increasing concentration of PSP, especially at a concentration of 4 mg/mL( P = 0.000). At 72 h, significant inhibition was observed at concentrations of 2 or 4 mg/mL༈ P = 0.000༉. As a result, PSP can effectively inhibit the growth of PC-3 cells. PSP induced the apoptosis of PC-3 cells and arrested cell cycle in S phase The PC-3 cells were treated with different concentrations of PSP for 72 h, and the apoptosis rate was analyzed by flow cytometry. The results showed that the apoptosis rate was significantly higher in the PSP group than in the blank control in a concentration-dependent manner ( P = 0.000, Fig. 2 A and Fig. 2 B). As we know, activated caspase-3 is generally considered a specific marker of apoptosis. In order to explore whether caspase-3 activation contributes to PSP-induced apoptosis, the activities of caspase-3 were detected by western blot after the treatment of PC-3 cells with PSP for 72 h. In this experiment, the concentration of PSP was selected as 0 µg/ml, 500 µg/ml, 1 mg/ml, 2 mg/ml and 4 mg/ml based on the results of apoptosis. The results indicated a concentration-dependent increase in the activities of caspase-3, suggesting the involvement of the intrinsic apoptotic pathway in PSP-induced apoptosis in PC-3 cells (Fig. 2 C). Cell cycle analysis showed that the cell percentage markedly increased in the S phase after 72 h of PSP treatment compared with the blank control. At a concentration of 4 mg/ml, PSP significantly increased the portion of PC-3 cells in the S phase from 19.7–49.0% (Fig. 3 ). These results indicated that PSP significantly promotes the apoptosis of PC-3 cells and arrests the cell cycle in the S phase. Moreover, PSP could induce the apoptosis of PC-3 cells in vitro through the caspase-3 pathway. PSP inhibited the migration and invasion of PC-3 cells To evaluate the effects of PSP on the invasiveness and migratory abilities of PC-3 cells. The PC-3 cells were treated with different concentrations of PSP for 72 h, and the invasion and migration of PC-3 cells were detected by the Transwell assay. As a result, the invasive and migratory cell numbers were significantly reduced with the increased concentration of PSP, especially at concentrations of 2 or 4 mg/ml (Fig. 4 ). PSP decreased the expression of the MDR-1 gene and its encoding protein, P-gp, in PC-3 cells. Studies have shown that high expression of MDR is associated with hormone-independent prostate cancer, so seeking effective drugs to reverse MDR is important to improve the efficacy of prostate cancer chemotherapy. As known, PC-3 cells are androgen-independent cells. In our study, human prostate cancer PC-3 cells were used as an MDR cell model to explore the expression of the MDR-1 gene and P-gp protein in PC-3 cells treated with PSP by real-time PCR and immunohistochemistry. The results indicated that MDR-1 gene expression decreased gradually with the increase in PSP concentrations, especially at concentrations of 4 mg/ml; MDR-1 gene expression was significantly lower than at other concentrations (Fig. 5 A). Similarly, the expression of P-gp also decreased gradually with the increase in PSP concentrations, especially at concentrations of 4 mg/mL (Fig. 5 B). All of these results showed that PSP might reverse the multidrug resistance in PC-3 cells. Reduced expression of p-P65, p-PI3K and p-AKT in PC-3 cells after treated with PSP To explore the potential mechanisms by which PSP influences the biological behavior of prostate cancer cells, proteins in the PI3K-AKT and NF-kB signaling pathway were analyzed by western blot after PC-3 cells were treated with PSP for 72 h. Results showed that AKT, PI3K, and P65 have no significant difference among groups, while phosphorylated P65, PI3K, and AKT (p-P65, p-PI3K,p-AKT) decreased in concentration-dependent manner, especially at concentration of 4 mg/ml. p-P65, p-PI3K, and p-AKT were significantly lower than those of other groups ( P = 0.000 ) (Fig. 6 A, 6 B). Discussion Nowadays, PSP has become more and more popular due to its potential applications in functional foods and medicine fields, 22 especially in the treatment of cancer. In this study, we investigated the function of PSP on prostate cancer PC-3 cells and initially explored the potential mechanism. We found that PSP dose-dependently inhibit the proliferation and induces the apoptosis of PC-3 cells. Our results are similar to those of one study which showed the inhibitory effect of PSP on HepG2 cells and the effect of inducing apoptosis on HepG2 cells 14 . While our study showed that PSP induces PC-3 cell cycle arrest in the S phase, which is different from the study that found PSP induces HepG2 cell cycle arrest in the G1 phase, this may be associated with different types of cells. Several studies showed PSP could stop mouse hepatoma cells H22 cells in the G0/G1 stage while holding human esophageal cancer ECA-109 cells, human gastric cancer HGC-27 cells, and human colorectal cancer HCT-8 cells in the S stage 5 , 15 , promoting their apoptosis. In addition, we found PSP dose-dependently inhibited the invasion and migration of PC-3 cells, and similar results were also found in another study that showed PSP dose-dependently inhibited migration, invasion, and epithelial-to-mesenchymal transition (EMT) of liver cancer cells. This study indicated that PSP dose-dependently reduced the activation of the Toll-like receptor 4 (TLR4)/Signal transducer and activator of transcription 3 (STAT3) and noncanonical nuclear factor-kappa B (NF-κB) signaling pathways, the author of this study considered that PSP inhibit liver cancer by eliminating the TLR4/STAT3 pathway 12 . In our study, we also explore the possible mechanisms of PSP-mediated apoptosis, migration, and invasion of prostate cancer cells. One study of how AKT/PKB expression correlates with the gleason pattern in human prostate cancer showed that AKT is upregulated in prostate cancer and that expression is correlated with tumor progression 23 . Another study of detecting Akt expression by immunohistochemical staining of paraffin-embedded tissue showed that the staining intensity for phosphor-Akt (p-AKT) was significantly greater in Gleason grades 8–10 compared with prostatic intraepithelial neoplasia and all other grades of prostate cancer 24 . That means, Akt and its related signaling pathway may participate in the progression of prostate cancer. So in our study, the expression of AKT and p-AKT were detected after PC-3 cells were treated with different doses of PSP. We found that AKT expression had no significant difference among each dose group, while p-AKT expression decreased with the increasing doses of PSP. As is known, protein kinase AKT is the dominate effector of PI3K signaling 25 . We then detected the expression of PI3K and p-PI3K. We also found that PI3K expression had no significant difference among each dose group, while p-PI3K expression decreased with increasing doses of PSP. Thus, it is speculated that the PI3K/Akt pathway might regulate the initiation and progression of prostate cancer, and PSP could inhibit the phosphorylation of AKT and PI3K to block prostate cancer cell proliferation, migration, and invasion. This is similar to another study, which indicated that palmitic acid inhibited the key molecules of the PI3K/Akt pathway to block prostate cancer proliferation and metastasis 26 . Additionally, one study showed that phosphorylation of NF-kB p65 ser536 plays a critical role in promoting prostate cancer oncogenesis, 27 this study also demonstrated the synergistic activities of NF-kB and AKT signals in promoting prostate cancer tumorigenesis 27 . In our study, we also found that P65 expressed in prostate cancer cells did not change after the treatment of PSP, while phosphorylated P65 (p-P65) decreased with increasing doses of PSP, that PSP may also inhibit the phosphorylation of P65 to block prostate cancer cells proliferation, migration and invasion. so, we thought PSP could inhibit he proliferation, invasion and migration of prostate cancer cells by regulating the PI3K/Akt and NF-κB signaling pathway. This exactly echoed the study which suggested the synergistic anticancer effect of salinomycin combined with cabazitaxel by downregulating Wnt, NF-kB and AKT signaling simultaneously 28 . Another study also demonstrated the functional link between PI3K-AKT and NF-kB pathways in the modulation of anti- apoptotic and multi drug resistant effects (the expression of MDR1 gene) in AML HL-60 cells 29 . In addition, multi-drug resistance in cancer cells significantly hinders the therapeutic efficacy of drugs 30 . Now, there is increasing interest in developing various therapeutic regimens, including some inhibitory drugs, to overcome MDR is increasing. Studies indicated natural products can impact multiple targets, which can be valuable in overcoming drug resistance from different perspectives 31 . Our study found that PSP, as a traditional Chinese medicine, can decrease the expression of the MDR-1 gene and its encoding protein P-gp, which might reverse the multidrug resistance in PC-3 cells. This may be associated with its anti-cancer effect. In summary, our present study showed for the first time that PSP could inhibit the proliferation, invasion, and migration of PC-3 cells in vitro , reverse the multidrug resistance in PC-3 cells. The possible mechanism is to regulate the PI3K/Akt and NF-kB signaling pathways simultaneously. However, our shortcomings are that we detected the function of PSP only on PC-3 cells. Does this conclusion apply to other prostate cancer cells, such as LNCap or DU145, or does it apply to the benign prostate hyperplasia cell lines? Next, we will further select more cell lines to detect the effect of PSP, and further investigation will also be conducted in mice. Declarations Significance Our findings suggest the antitumor potential of PSP for prostate cancer by targeting the PI3K/Akt and NF-kB pathways. Competing interests: The author declares no conflict of interest. Funding: This work was supported by the Natural Science Foundation of Hebei Province (H2021405012). Author Contribution Conceptualization, G.Z. and Y.Z.; methodology, Y.T.,S.D., X.Z. and X.L.; software, X.W., C.L. andS.D.; validation,Y.Z, Y.T., S.D., X.Z. and X.W.; formal analysis, Y.T., S.D. and C.L.; investigation, G.Z. and Y.Z.;resources, S.D., Y.T. and X.Z.; data curation, Y.T., Y.Z., X.W., X.L. and C.L.; writing—original draft preparation, Y.Z., X.Z., S.D. and G.Z.; writing-review and editing,M.A.,Y.Z., X.W. and Y.T.; visualization,M.A.,G.Z. and Y.Z.;supervision, G.Z.; project administration, G.Z. All authors have read and agreed to the published version of the manuscript. Acknowledgement Conceptualization, G.Z. and Y.Z.; methodology, Y.T.,S.D., X.Z. and X.L.; software, X.W., C.L. andS.D.; validation,Y.Z, Y.T., S.D., X.Z. and X.W.; formal analysis, Y.T., S.D. and C.L.; investigation, G.Z. and Y.Z.;resources, S.D., Y.T. and X.Z.; data curation, Y.T., Y.Z., X.W., X.L. and C.L.; writing—original draft preparation, Y.Z., X.Z., S.D. and G.Z.; writing-review and editing,M.A.,Y.Z., X.W. and Y.T.; visualization,M.A.,G.Z. and Y.Z.;supervision, G.Z.; project administration, G.Z. All authors have read and agreed to the published version of the manuscript. Data availability: Data sharing not applicable to this article as no datasets were generated or analysed during the current study. References Silk N, Reich J, Sinha R, et al. The Effects of Resveratrol on Prostate Cancer through Targeting the Tumor Microenvironment. J Xenobiot. 2021 Feb 1;11(1):16-32. PMID: 33535458. Penezić L, Kuliš T, Hudolin T, et al. LAPAROSCOPIC RADICAL PROSTATECTOMY: SINGLE CENTER CASE SERIES. Acta Clin Croat. 2022 Oct;61(Suppl 3):15-20. PMID: 36938548. Stoykova GE, Schlaepfer IR. Lipid Metabolism and Endocrine Resistance in Prostate Cancer, and New Opportunities for Therapy. Int J Mol Sci. 2019 May 28;20(11):2626. PMID: 31142021. Cai M, Song XL, Li XA, et al. Current therapy and drug resistance in metastatic castration-resistant prostate cancer. Drug Resist Updat. 2023 May;68:100962.PMID: 37068396. Cui X, Wang S, Cao H, et al. A Review: The Bioactivities and Pharmacological Applications of Polygonatumsibiricum polysaccharides. Molecules. 2018 May 14;23(5):1170. PMID: 29757991. Zhang H, Li H, Pan B, et al. Integrated 16S rRNA Sequencing and Untargeted Metabolomics Analysis to Reveal the Protective Mechanisms of Polygonatum sibiricum Polysaccharide on Type 2 Diabetes Mellitus Model Rats.Curr Drug Metab. 2023;24(4):270-282. PMID: 37038712. Wang Y, Lan C, Liao X, et al. Polygonatum sibiricum polysaccharide potentially attenuates diabetic retinal injury in a diabetic rat model. J Diabetes Investig. 2019 Jul;10(4):915-924. PMID: 30426692. Yang JX, Wu S, Huang XL, et al. Hypolipidemic Activity and Anti-atherosclerotic Effect of Polysaccharide of Polygonatum sibiricum in Rabbit Model and Related Cellular Mechanisms. Evid Based Complement Alternat Med. 2015;2015:391065. PMID: 26089938. Ye G, Zhao Y, Zhu J, et al. Synergistic Effect of Polydatin and Polygonatum sibiricum Polysaccharides in Combating Atherosclerosis via Suppressing TLR4-Mediated NF- κ B Activation in ApoE-Deficient Mice. Evid Based Complement Alternat Med. PMID: 35845572. Du L, Nong MN, Zhao JM, et al. Polygonatum sibiricum polysaccharide inhibits osteoporosis by promoting osteoblast formation and blocking osteoclastogenesis through Wnt/β-catenin signaling pathway. Sci Rep. 2016 Aug 24;6:32261. PMID: 27554324. Long T, Liu Z, Shang J, et al.Polygonatum sibiricum polysaccharides play anti-cancer effect through TLR4-MAPK/NF-κB signaling pathways. Int J Biol Macromol. 2018 May;111:813-821.PMID: 29343453. Xu Y, Guo Y, Lu C, et al.Polygonatum sibiricum polysaccharide Inhibited Liver Cancer in a Simulated Tumor Microenvironment by Eliminating TLR4/STAT3 Pathway. Biol Pharm Bull. 2023;46(9):1249-1259.PMID: 37661404. Han SY, Hu MH，Qi GY，et al. Polysaccharides from Polygonatum Inhibit the Proliferation of Prostate Cancer-Associated Fibroblasts. Asian Pac J Cancer Prev. 2016;17(8):3829-33. PMID: 27644624. Li M, Liu Y, Zhang H, et al. Anti-cancer Potential of Polysaccharide Extracted From Polygonatum sibiricum on HepG2 Cells via Cell Cycle Arrest and Apoptosis. Front Nutr. 2022 Jul 4;9:938290. PMID: 35903453. Li L, Tian LN, Ren ZX, et al. Research progress on the structural analysis and functional activity of polysaccharides. Chin. J. Exp. Tradit. Med. Fromul. 2015, 21,231–234. DOI:10.13422/j.cnki.syfjx.2015150231(in Chinese). Tewari D, Patni P, Bishayee A, et al. Natural products targeting the PI3K-Akt-mTOR signaling pathway in cancer: A novel therapeutic strategy. Semin Cancer Biol. 2022 May;80:1-17.PMID: 31866476. Hashemi M, Taheriazam A, Daneii P, et al. Targeting PI3K/Akt signaling in prostate cancer therapy. J Cell Commun Signal. 2023 Sep;17(3):423-443. PMID: 36367667. Wang Q, Wu S, Gu Y, et al.RASAL2 regulates the cell cycle and cyclin D1 expression through PI3K/AKT signalling in prostate tumorigenesis. Cell Death Discov. 2022 Jun 6;8(1):275. PMID: 35668070. Gu Y, Liang C. TRAIP suppressed apoptosis and cell cycle to promote prostate cancer proliferation via TRAF2-PI3K-AKT pathway activation. Int Urol Nephrol. 2023 Dec 15. PMID: 38100027. Raja Singh P, Sugantha Priya E, Balakrishnan S, et al. Inhibition of cell survival and proliferation by nimbolide in human androgen-independent prostate cancer (PC-3) cells: involvement of the PI3K/Akt pathway. Mol Cell Biochem. 2017 Mar;427(1-2):69-79. PMID: 28025797. Chen H, Zhou L, Wu X, et al. The PI3K/AKT pathway in the pathogenesis of prostate cancer. Front Biosci (Landmark Ed). 2016 Jun 1;21(5):1084-91. PMID: 27100493. Liu D, Tang W, Han C, et al. Advances in Polygonatum sibiricum polysaccharides: Extraction, purification, structure, biosynthesis, and bioactivity. Front Nutr. 2022 Dec 5;9:1074671. PMID: 36545471. Liao YD, Grobholz R, Abel U, et al. Increase of AKT/PKB expression correlates with gleason pattern in human prostate cancer. Int J Cancer. 2003 Nov 20;107(4):676-80. PMID: 14520710. Malik SN, Brattain M, Ghosh PM, Immunohistochemical demonstration of phospho-Akt in high Gleason grade prostate cancer. Clin Cancer Res. 2002 Apr;8(4):1168-71.PMID: 11948129. Lien EC, Dibble CC, Toker A. PI3K signaling in cancer: beyond AKT. CurrOpin Cell Biol. 2017 Apr;45:62-71. PMID: 28343126. Zhu S, Jiao W, Xu Y, et al. Palmitic acid inhibits prostate cancer cell proliferation and metastasis by suppressing the PI3K/Akt pathway. Life Sci. 2021 Dec 1;286:120046. PMID: 34653428. Zhang L, Shao L, Creighton CJ, et al. Function of phosphorylation of NF-kB p65 ser536 in prostate cancer oncogenesis. Oncotarget. 2015 Mar 20;6(8):6281-94. PMID: 25749044. Erdogan S, Serttas R, Turkekul K, et al. The synergistic anticancer effect of salinomycin combined with cabazitaxel in CD44 + prostate cancer cells by downregulating wnt, NF-κB and AKT signaling. Mol Biol Rep. 2022 Jun;49(6):4873-4884. PMID: 35705771. Davoudi Z, Akbarzadeh A, Rahmatiyamchi M, et al. Molecular Target Therapy of AKT and NF-kB Signaling Pathways and Multidrug Resistance by Specific Cell Penetrating Inhibitor Peptides in HL-60 Cells. Asian Pac J Cancer Prev. 2014;15(10):4353-8. PMID: 24935396. Duan C, Yu M, Xu J, et al. Overcoming Cancer Multi-drug Resistance (MDR): Reasons, mechanisms, nanotherapeutic solutions, and challenges. Biomed Pharmacother. 2023 Jun;162:114643. PMID: 37031496. Chen T, Xiao Z, Liu X, et al. Natural products for combating multidrug resistance in cancer. Pharmacol Res. 2024 Apr;202:107099. PMID: 38342327. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editor assigned by journal 04 Jul, 2024 Submission checks completed at journal 03 Jul, 2024 First submitted to journal 01 Jul, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4668118","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":322632645,"identity":"05241d92-34fa-4be4-8eea-40fe795944da","order_by":0,"name":"Guobin Zhao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwElEQVRIie3RoQ7CMBCA4WuWFNNttoryCCNYHqYYZoavJ6lamJ0g8ARYdJsmqO4JMCRYQiYn6RRyJ0nor07cJ64FiMV+MgamV3xOZ3uDJsS2fr3K2E2iSeJSvd2ceLXAAdFcjGPelRoqgEFdpwlp39Ieldtp6Ayp/X2aJNwX5uUDIQeZEI0gdCSpdiVNWIEjLK8LG86XlGIJ55kcH3mpGZUWdYtoOteHrxTi/LSPQSEIcPmdDWI/lCP3YrFY7I/7AD48QSoqd2ssAAAAAElFTkSuQmCC","orcid":"","institution":"The First Affiliated Hospital of Hebei North University","correspondingAuthor":true,"prefix":"","firstName":"Guobin","middleName":"","lastName":"Zhao","suffix":""},{"id":322632646,"identity":"497e4820-3389-4b7c-809f-3a1f219b3c81","order_by":1,"name":"Yan Zhou","email":"","orcid":"","institution":"Laboratory Medicine College of Hebei North University","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Zhou","suffix":""},{"id":322632647,"identity":"935fda8f-eb67-4c93-bd38-808ea0f44148","order_by":2,"name":"Yuhong Tang","email":"","orcid":"","institution":"Laboratory Medicine College of Hebei North University","correspondingAuthor":false,"prefix":"","firstName":"Yuhong","middleName":"","lastName":"Tang","suffix":""},{"id":322632648,"identity":"58026899-231f-41b2-813a-04e09f2ab3a2","order_by":3,"name":"Muhammad Abbas","email":"","orcid":"","institution":"The First Affiliated Hospital of Hebei North University","correspondingAuthor":false,"prefix":"","firstName":"Muhammad","middleName":"","lastName":"Abbas","suffix":""},{"id":322632649,"identity":"c987aa79-9e76-4b63-87ea-1d059b5e28a1","order_by":4,"name":"Shaowen Dong","email":"","orcid":"","institution":"The First Affiliated Hospital of Hebei North University","correspondingAuthor":false,"prefix":"","firstName":"Shaowen","middleName":"","lastName":"Dong","suffix":""},{"id":322632650,"identity":"d305923c-5049-4ea0-a08f-b82553dcc5a4","order_by":5,"name":"Xinyang Zhao","email":"","orcid":"","institution":"The First Affiliated Hospital of Hebei North University","correspondingAuthor":false,"prefix":"","firstName":"Xinyang","middleName":"","lastName":"Zhao","suffix":""},{"id":322632651,"identity":"3bfb8f5d-ee62-4145-bbdd-488a43cb2790","order_by":6,"name":"Xin Liu","email":"","orcid":"","institution":"The First Affiliated Hospital of Hebei North University","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Liu","suffix":""},{"id":322632652,"identity":"5a9b3c25-53d8-4b9f-8a30-0306a4e7c149","order_by":7,"name":"Xinmei Wang","email":"","orcid":"","institution":"The First Affiliated Hospital of Hebei North University","correspondingAuthor":false,"prefix":"","firstName":"Xinmei","middleName":"","lastName":"Wang","suffix":""},{"id":322632653,"identity":"8a1c798a-6906-425e-b309-f0aae476514c","order_by":8,"name":"Chen Li","email":"","orcid":"","institution":"The First Affiliated Hospital of Hebei North University","correspondingAuthor":false,"prefix":"","firstName":"Chen","middleName":"","lastName":"Li","suffix":""},{"id":322632654,"identity":"a90d855e-7a9e-4ff6-8804-33ff86c38785","order_by":9,"name":"Chenxi Liu","email":"","orcid":"","institution":"The Graduate School of Hebei North University","correspondingAuthor":false,"prefix":"","firstName":"Chenxi","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2024-07-01 12:42:56","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4668118/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4668118/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":61320793,"identity":"89ec8aac-597e-48e5-9468-aeee41377268","added_by":"auto","created_at":"2024-07-29 13:03:38","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":76782,"visible":true,"origin":"","legend":"\u003cp\u003ePSP inhibited cell proliferation in PC-3 cells. PC-3 cells were seeded into 96-well tissue plates and incubated with various concentrations of PSP for 24 h, 48 h, and 72 h. The CCK-8 assay was used to detect the proliferation of cells. PSP could effectively inhibit the growth of PC-3 cells at concentrations of 2 or 4 mg/mL for 48h and 72h. (** \u003cem\u003eP \u003c/em\u003e\u0026lt;0.01).\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4668118/v1/27b88c3705aa7710b4e465ca.jpeg"},{"id":61321392,"identity":"b717a05d-4f9d-4a75-b647-c9fff16fe36c","added_by":"auto","created_at":"2024-07-29 13:11:38","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":395590,"visible":true,"origin":"","legend":"\u003cp\u003ePSP could promote cell apoptosis and expression of caspase-3. PC-3 cells were treated with different concentrations of PSP for 72 h, the cell apoptosis was analyzed by flow cytometry(A) and the apoptotic rateswas shown in column(B). The treated cell lysate was collected and the expression of caspase-3 was detected by western blot (C). （** \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01）\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4668118/v1/0edbc46a873a62c3851b2d93.jpeg"},{"id":61320789,"identity":"9df076c1-90f8-4ca2-b7ea-c087c7a8acc3","added_by":"auto","created_at":"2024-07-29 13:03:38","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":213464,"visible":true,"origin":"","legend":"\u003cp\u003ePSP could arrest cell cycle of PC-3 cells in S phase. PC-3 Cells were treated with various concentrations of PSP for 72 h and harvested. Cell cycle progression was assayed using propidium iodide staining detected by flow cytometry.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4668118/v1/7071a1992d3e81393698482d.jpeg"},{"id":61320795,"identity":"f70ec6cf-a8ab-454f-83db-af490192bda1","added_by":"auto","created_at":"2024-07-29 13:03:38","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1015591,"visible":true,"origin":"","legend":"\u003cp\u003ePSP suppressed PC-3 cell migration (A) and invasiveness (B) in vitro. PC-3 cells were treated with different concentrations of PSP for 72 h, then seeded in 24-well plates coated with or without Matrigel in the upper compartment and the lower compartment. After 24 h of incubation, migrated or invaded cells were stained by Giemsa. The migratory and invasive PC-3 cell numbers were reduced with the increased concentration of PSP.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4668118/v1/65f2cf1e8be5447b2ccd5e99.jpeg"},{"id":61321393,"identity":"e9d255dd-55a2-4c32-b472-ac76f2da97ab","added_by":"auto","created_at":"2024-07-29 13:11:38","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":521446,"visible":true,"origin":"","legend":"\u003cp\u003ePSP decreased the expression of the MDR-1 gene and P-gp protein in PC-3 cells. PC-3 cells were treated with different concentrations of PSP for 72 h, total RNA was extracted and reverse transcribed into cDNA, and then real-time PCR was used to detect the expression of the MDR-1 gene(A). The treated PC-3 cell slides were prepared, and immunohistochemistry was used to detect the expression of P-gp(B). （** \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01）\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4668118/v1/7798fce6e451411997a2548c.jpeg"},{"id":61320791,"identity":"c667a88e-3b6c-4610-9e8c-dcdaf04e1e1b","added_by":"auto","created_at":"2024-07-29 13:03:38","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":328525,"visible":true,"origin":"","legend":"\u003cp\u003eThe protein expression(A) and the relative quantification analysis(B) of AKT, p-AKT, PI3K, p-PI3K, P65, and p-P65. PC-3 cells treated with various concentrations of PSP were harvested and lysed with RIPA. After quantification with BCA, SDS-PAGE gel electrophoresis was performed at 50 ug of protein per well, and then Western blot was used to analyze the protein expression. The results indicated that the phosphorylated P65, PI3K, and AKT (p-P65, p-PI3K, p-AKT) decreased in concentration-dependent manner, especially at concentration of 4 mg/ml (** \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01)(B). The samples derive from the same experiment, and the blots were processed in parallel.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4668118/v1/af40899b18e803820153f1fa.jpeg"},{"id":61322258,"identity":"99adf5ef-115a-4655-b81e-f5a9191d20ff","added_by":"auto","created_at":"2024-07-29 13:19:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3006210,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4668118/v1/043c9ff7-a850-49a9-b411-8cafd4629031.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The potential antitumor activity of polysaccharide extracted from polygonatumsibiricum on human prostate cancer PC-3 cells ","fulltext":[{"header":"Introduction","content":"\u003cp\u003eProstate cancer is the second-leading cause of cancer-related deaths in men worldwide\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. For early-stage prostate cancer, laparoscopic radical prostatectomy can achieve a radical treatment effect that has the characteristics of less expensive, minimally invasive\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e and quick recovery after operation. For relapse or metastatic prostate cancer, radical surgery is not suitable, while Androgen deprivation therapy (ADT) is the standard-of-care therapy at present\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, aiming at removing androgens and inhibiting the activity of the androgens to delay the progression of prostate cancer. However, most prostate cancer patients might gradually become insensitive or ineffective to androgen blocking, thus developing castration-resistant prostate cancer (CRPC) with high morbidity and mortality\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. It is very difficult for clinical treatment because of the natural or acquired drug resistance of CRPC, and the prognosis is poor. Therefore, it is necessary to find a new approach to effectively treat these patients with advanced prostate cancer.\u003c/p\u003e \u003cp\u003eRecently, many studies have shown that polygonatum sibiricum polysaccharides(PSP) have many pharmacological applications and biological activities\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e and are widely used in treatments against diabetes mellitus and its complications\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, Hypoplipidemic\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, anti-atherosclerosis\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e,\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, anti-osteoporosis\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e and cancers\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Of these, the possible anti-tumor effect of PSP has attracted the interest of scientists because it is less toxic and has no side effects. One study indicated that PSP could significantly inhibit the growth of prostate-CAF (cancer-associated fibroblasts and may improve the efficacy of cancer therapy\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Another study showed that PSP could induce HepG2 cell apoptosis, and arresting the cell cycle at the G1 phase might have potential antitumor effects\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Several studies also suggested that PSP could hold human gastric cancer HGC-27 cells, esophageal cancer ECA-109 cells, and colorectal cancer HCT-8 cells in the S stage, promoting their apoptosis\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. How PSP influenced the prostate cancer PC-3 cell cycle and induced apoptosis? It is necessary to further explore the anti-tumor mechanisms.\u003c/p\u003e \u003cp\u003eStudies have indicated that phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/AKT) signaling pathway is one of the key signaling pathways involved in the regulation of cell cycle\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Most studies also found that PI3K/AKT signaling plays a significant role in prostate cancer tumorigenesis and therapy, which participate in the apoptosis, proliferation, metastasis, and invasion of prostate cells\u003csup\u003e\u003cspan additionalcitationids=\"CR18 CR19 CR20\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSo, our hypothesis is that whether PSP affects the biological behavior of prostate cancer cells and whether the mechanism is related to the PI3K signaling pathway. In this study, we will explore the function of PSP on prostate cancer PC-3 cells and elucidate its possible mechanism.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell culture and reagents\u003c/h2\u003e \u003cp\u003eHuman prostate cancer PC-3 cells were purchased from Wuhan Procell Life Science \u0026amp; Technology Co., Ltd. Cells were cultured in Ham's F-12K supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 mg/ml streptomycin at 37\u0026deg;C in a humidified atmosphere containing 5% CO2. PSP was provided by Yuanye Biotechnology Co., Ltd. The compounds were dissolved in PBS to 20 mg/ml as a stock solution, stored at -20\u0026deg;C for in vitro studies, and diluted wit\u003c/p\u003e \u003cp\u003eh medium before each assay.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCell Counting kit-8(CCK-8) assay\u003c/h2\u003e \u003cp\u003eThe effect of PSP on cell growth was determined using the CCK-8 assay. Cells were seeded into 96-well tissue plates at a density of 1\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/well and incubated with various concentrations of PSP for 24 h, 48 h, or 72 h. Untreated cells served as the control group. After treatment, 10\u0026micro;l of CCK-8 reagent was added to each well and then incubated for an additional 4 hours at 37\u0026deg;C. The cell viability was determined by measuring the absorbance at 450 nm using a microplate reader.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry\u003c/h2\u003e \u003cp\u003eBased on the results of the CCK-8 assay, PC-3 cells were plated into 6-well plates (2\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well) and treated with various concentrations of PSP solution for 72 h.\u003c/p\u003e \u003cp\u003eThe apoptosis rate of PC-3 cells was evaluated using the Annexin V-FITC/PI Apoptosis Detection Kit according to the instructions from the manufacturer. The cultured cells were collected, washed with cold PBS, and resuspended in binding buffer. 5 \u0026micro;l of Annexin V-FITC and 5 \u0026micro;l of PI were added to the buffer and incubated at room temperature for 15 min in the dark. Annexin V-FITC binds to phosphatidylserine on the outer membrane of apoptotic cells, while PI enters and stains cells with compromised membrane integrity and then binds to and labels DNA. A flow cytometer (BEAMDIAG; BeamCyte-1026, China) was used for data collection, and FlowJo software for data analysis.\u003c/p\u003e \u003cp\u003eFor the cell cycle assay, PC-3 cells were digested by trypsin, washed with cold PBS, and fixed with 70% alcohol at 4\u0026deg;C overnight. Subsequently, the fixed cells were collected and resuspended in cold PBS, then the cells were stained with PI and incubated for 30 min at 37\u0026deg;C in the dark. A flow cytometer (BEAMDIAG; BeamCyte-1026, China) was used for data collection, and FlowJo software for data analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eTranswell assay\u003c/h2\u003e \u003cp\u003eA Transwell assay was used to analyze cell invasion and/or migration. Cells treated with PSP suspended in FBS-free medium were seeded in 24-well plates coated with or without Matrigel in the upper compartment, and the lower compartment was supplemented with 10% FBS. After 24 hours of incubation, non-invaded or non-migrated cells across the membranes were carefully removed with cotton swabs, while cells across the membrane were fixed with carbinol and then stained using Giemsa. After that, cells were calculated under a microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot\u003c/h2\u003e \u003cp\u003eCells treated with PSP were harvested and lysed with RIPA, and protein levels were measured with a BCA quantification kit. The lysates were subjected to 10% SDS-PAGE and transferred to the PVDF membrane. Membranes were probed at 4\u0026deg;C in the presence of 1:1000 dilutions of primary antibodies, including Akt, p-Akt, PI3K, p-PI3K, caspase-3, P65, and p-P65. After being washed three times with PBS-T, the membranes were incubated with HRP-labelled sheep anti-mouse antibody for 1 h. Finally, the blots were visualized with enhanced chemiluminescence.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eReal-time RT-PCR\u003c/h2\u003e \u003cp\u003ePC-3 cells were treated with different concentrations of PSP for 72 hours, then the total RNA was extracted using a Trizol reagent, and the RNA concentration was determined using NanoDrop 2000. 1 \u0026micro;g of RNA was reverse transcribed into cDNA with the SuperRT cDNA Synthesis Kit. GAPDH was used as an internal control. Levels of MDR1 mRNA were assessed using the UltraSYBR Mixture (Low ROX) (Beijing Comwin Biotech Co., Ltd.). The data were normalized relative to GAPDH by using the following formula: relative mRNA expression\u0026thinsp;=\u0026thinsp;2-△△Ct, where Ct is the cycle threshold.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry (IHC)\u003c/h2\u003e \u003cp\u003ePSP-treated cell slides were prepared, followed by serum blocking for 30 minutes at 37\u0026deg;C and staining with anti-MDR1/P-gp antibodies overnight at 4\u0026deg;C. Then the slides were incubated with HRP-conjugated secondary anti-rabbit IgG at 37\u0026deg;C for 40 minutes. Slides were visualized with diaminobenzidine (DAB), counterstained with hematoxylin, and observed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll experiments were conducted three times. The data were analyzed by SPSS 19.0 statistical software. The experimental results were expressed as the mean standard deviation. The difference was \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePSP inhibited the proliferation of PC-3 cells\u003c/h2\u003e \u003cp\u003eThe CCK-8 assay was used to investigate whether PSP inhibits the growth of PC-3 cells at the appropriate inhibitory concentration. PC-3 cells were exposed to various concentrations of PSP (0 ug/mL, 125 ug/mL, 250 ug/mL, 500 ug/mL, 1 mg/mL, 2 mg/mL, and 4 mg/mL) for 24 h, 48 h, and 72 h. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, At 24 h, no obvious inhibition was observed at different concentrations. At 48 h, the inhibition increased with the increasing concentration of PSP, especially at a concentration of 4 mg/mL(\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.000). At 72 h, significant inhibition was observed at concentrations of 2 or 4 mg/mL༈\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.000༉. As a result, PSP can effectively inhibit the growth of PC-3 cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ePSP induced the apoptosis of PC-3 cells and arrested cell cycle in S phase\u003c/h2\u003e \u003cp\u003eThe PC-3 cells were treated with different concentrations of PSP for 72 h, and the apoptosis rate was analyzed by flow cytometry. The results showed that the apoptosis rate was significantly higher in the PSP group than in the blank control in a concentration-dependent manner (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.000, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eAs we know, activated caspase-3 is generally considered a specific marker of apoptosis. In order to explore whether caspase-3 activation contributes to PSP-induced apoptosis, the activities of caspase-3 were detected by western blot after the treatment of PC-3 cells with PSP for 72 h. In this experiment, the concentration of PSP was selected as 0 \u0026micro;g/ml, 500 \u0026micro;g/ml, 1 mg/ml, 2 mg/ml and 4 mg/ml based on the results of apoptosis. The results indicated a concentration-dependent increase in the activities of caspase-3, suggesting the involvement of the intrinsic apoptotic pathway in PSP-induced apoptosis in PC-3 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eCell cycle analysis showed that the cell percentage markedly increased in the S phase after 72 h of PSP treatment compared with the blank control. At a concentration of 4 mg/ml, PSP significantly increased the portion of PC-3 cells in the S phase from 19.7\u0026ndash;49.0% (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThese results indicated that PSP significantly promotes the apoptosis of PC-3 cells and arrests the cell cycle in the S phase. Moreover, PSP could induce the apoptosis of PC-3 cells in vitro through the caspase-3 pathway.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePSP inhibited the migration and invasion of PC-3 cells\u003c/h2\u003e \u003cp\u003eTo evaluate the effects of PSP on the invasiveness and migratory abilities of PC-3 cells. The PC-3 cells were treated with different concentrations of PSP for 72 h, and the invasion and migration of PC-3 cells were detected by the Transwell assay. As a result, the invasive and migratory cell numbers were significantly reduced with the increased concentration of PSP, especially at concentrations of 2 or 4 mg/ml (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003ePSP decreased the expression of the MDR-1 gene and its encoding protein, P-gp, in PC-3 cells.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eStudies have shown that high expression of MDR is associated with hormone-independent prostate cancer, so seeking effective drugs to reverse MDR is important to improve the efficacy of prostate cancer chemotherapy. As known, PC-3 cells are androgen-independent cells. In our study, human prostate cancer PC-3 cells were used as an MDR cell model to explore the expression of the MDR-1 gene and P-gp protein in PC-3 cells treated with PSP by real-time PCR and immunohistochemistry. The results indicated that MDR-1 gene expression decreased gradually with the increase in PSP concentrations, especially at concentrations of 4 mg/ml; MDR-1 gene expression was significantly lower than at other concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Similarly, the expression of P-gp also decreased gradually with the increase in PSP concentrations, especially at concentrations of 4 mg/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). All of these results showed that PSP might reverse the multidrug resistance in PC-3 cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eReduced expression of p-P65, p-PI3K and p-AKT in PC-3 cells after treated with PSP\u003c/h2\u003e \u003cp\u003eTo explore the potential mechanisms by which PSP influences the biological behavior of prostate cancer cells, proteins in the PI3K-AKT and NF-kB signaling pathway were analyzed by western blot after PC-3 cells were treated with PSP for 72 h. Results showed that AKT, PI3K, and P65 have no significant difference among groups, while phosphorylated P65, PI3K, and AKT (p-P65, p-PI3K,p-AKT) decreased in concentration-dependent manner, especially at concentration of 4 mg/ml. p-P65, p-PI3K, and p-AKT were significantly lower than those of other groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.000 ) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eNowadays, PSP has become more and more popular due to its potential applications in functional foods and medicine fields,\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e especially in the treatment of cancer. In this study, we investigated the function of PSP on prostate cancer PC-3 cells and initially explored the potential mechanism. We found that PSP dose-dependently inhibit the proliferation and induces the apoptosis of PC-3 cells. Our results are similar to those of one study which showed the inhibitory effect of PSP on HepG2 cells and the effect of inducing apoptosis on HepG2 cells\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. While our study showed that PSP induces PC-3 cell cycle arrest in the S phase, which is different from the study that found PSP induces HepG2 cell cycle arrest in the G1 phase, this may be associated with different types of cells. Several studies showed PSP could stop mouse hepatoma cells H22 cells in the G0/G1 stage while holding human esophageal cancer ECA-109 cells, human gastric cancer HGC-27 cells, and human colorectal cancer HCT-8 cells in the S stage\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, promoting their apoptosis.\u003c/p\u003e \u003cp\u003eIn addition, we found PSP dose-dependently inhibited the invasion and migration of PC-3 cells, and similar results were also found in another study that showed PSP dose-dependently inhibited migration, invasion, and epithelial-to-mesenchymal transition (EMT) of liver cancer cells. This study indicated that PSP dose-dependently reduced the activation of the Toll-like receptor 4 (TLR4)/Signal transducer and activator of transcription 3 (STAT3) and noncanonical nuclear factor-kappa B (NF-κB) signaling pathways, the author of this study considered that PSP inhibit liver cancer by eliminating the TLR4/STAT3 pathway\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn our study, we also explore the possible mechanisms of PSP-mediated apoptosis, migration, and invasion of prostate cancer cells. One study of how AKT/PKB expression correlates with the gleason pattern in human prostate cancer showed that AKT is upregulated in prostate cancer and that expression is correlated with tumor progression\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Another study of detecting Akt expression by immunohistochemical staining of paraffin-embedded tissue showed that the staining intensity for phosphor-Akt (p-AKT) was significantly greater in Gleason grades 8\u0026ndash;10 compared with prostatic intraepithelial neoplasia and all other grades of prostate cancer\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. That means, Akt and its related signaling pathway may participate in the progression of prostate cancer. So in our study, the expression of AKT and p-AKT were detected after PC-3 cells were treated with different doses of PSP. We found that AKT expression had no significant difference among each dose group, while p-AKT expression decreased with the increasing doses of PSP. As is known, protein kinase AKT is the dominate effector of PI3K signaling\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. We then detected the expression of PI3K and p-PI3K. We also found that PI3K expression had no significant difference among each dose group, while p-PI3K expression decreased with increasing doses of PSP. Thus, it is speculated that the PI3K/Akt pathway might regulate the initiation and progression of prostate cancer, and PSP could inhibit the phosphorylation of AKT and PI3K to block prostate cancer cell proliferation, migration, and invasion. This is similar to another study, which indicated that palmitic acid inhibited the key molecules of the PI3K/Akt pathway to block prostate cancer proliferation and metastasis\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Additionally, one study showed that phosphorylation of NF-kB p65 ser536 plays a critical role in promoting prostate cancer oncogenesis,\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e this study also demonstrated the synergistic activities of NF-kB and AKT signals in promoting prostate cancer tumorigenesis\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. In our study, we also found that P65 expressed in prostate cancer cells did not change after the treatment of PSP, while phosphorylated P65 (p-P65) decreased with increasing doses of PSP, that PSP may also inhibit the phosphorylation of P65 to block prostate cancer cells proliferation, migration and invasion. so, we thought PSP could inhibit he proliferation, invasion and migration of prostate cancer cells by regulating the PI3K/Akt and NF-κB signaling pathway. This exactly echoed the study which suggested the synergistic anticancer effect of salinomycin combined with cabazitaxel by downregulating Wnt, NF-kB and AKT signaling simultaneously\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Another study also demonstrated the functional link between PI3K-AKT and NF-kB pathways in the modulation of anti- apoptotic and multi drug resistant effects (the expression of MDR1 gene) in AML HL-60 cells\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn addition, multi-drug resistance in cancer cells significantly hinders the therapeutic efficacy of drugs\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Now, there is increasing interest in developing various therapeutic regimens, including some inhibitory drugs, to overcome MDR is increasing. Studies indicated natural products can impact multiple targets, which can be valuable in overcoming drug resistance from different perspectives\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Our study found that PSP, as a traditional Chinese medicine, can decrease the expression of the MDR-1 gene and its encoding protein P-gp, which might reverse the multidrug resistance in PC-3 cells. This may be associated with its anti-cancer effect.\u003c/p\u003e \u003cp\u003eIn summary, our present study showed for the first time that PSP could inhibit the proliferation, invasion, and migration of PC-3 cells in \u003cem\u003evitro\u003c/em\u003e, reverse the multidrug resistance in PC-3 cells. The possible mechanism is to regulate the PI3K/Akt and NF-kB signaling pathways simultaneously. However, our shortcomings are that we detected the function of PSP only on PC-3 cells. Does this conclusion apply to other prostate cancer cells, such as LNCap or DU145, or does it apply to the benign prostate hyperplasia cell lines? Next, we will further select more cell lines to detect the effect of PSP, and further investigation will also be conducted in mice.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eSignificance\u003c/strong\u003e \u003cp\u003eOur findings suggest the antitumor potential of PSP for prostate cancer by targeting the PI3K/Akt and NF-kB pathways.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompeting interests:\u003c/strong\u003e \u003cp\u003eThe author declares no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThis work was supported by the Natural Science Foundation of Hebei Province (H2021405012).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization, G.Z. and Y.Z.; methodology, Y.T.,S.D., X.Z. and X.L.; software, X.W., C.L. andS.D.; validation,Y.Z, Y.T., S.D., X.Z. and X.W.; formal analysis, Y.T., S.D. and C.L.; investigation, G.Z. and Y.Z.;resources, S.D., Y.T. and X.Z.; data curation, Y.T., Y.Z., X.W., X.L. and C.L.; writing\u0026mdash;original draft preparation, Y.Z., X.Z., S.D. and G.Z.; writing-review and editing,M.A.,Y.Z., X.W. and Y.T.; visualization,M.A.,G.Z. and Y.Z.;supervision, G.Z.; project administration, G.Z. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eConceptualization, G.Z. and Y.Z.; methodology, Y.T.,S.D., X.Z. and X.L.; software, X.W., C.L. andS.D.; validation,Y.Z, Y.T., S.D., X.Z. and X.W.; formal analysis, Y.T., S.D. and C.L.; investigation, G.Z. and Y.Z.;resources, S.D., Y.T. and X.Z.; data curation, Y.T., Y.Z., X.W., X.L. and C.L.; writing\u0026mdash;original draft preparation, Y.Z., X.Z., S.D. and G.Z.; writing-review and editing,M.A.,Y.Z., X.W. and Y.T.; visualization,M.A.,G.Z. and Y.Z.;supervision, G.Z.; project administration, G.Z. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eData availability:\u003c/h2\u003e \u003cp\u003eData sharing not applicable to this article as no datasets were generated or analysed during the current study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSilk N, Reich J, Sinha R, et al. The Effects of Resveratrol on Prostate Cancer through Targeting the Tumor Microenvironment. J Xenobiot. 2021 Feb 1;11(1):16-32. PMID: 33535458.\u003c/li\u003e\n\u003cli\u003ePenezić L, Kuli\u0026scaron; T, Hudolin T, et al. LAPAROSCOPIC RADICAL PROSTATECTOMY: SINGLE CENTER CASE SERIES. Acta Clin Croat. 2022 Oct;61(Suppl 3):15-20. PMID: 36938548.\u003c/li\u003e\n\u003cli\u003eStoykova GE, Schlaepfer IR. Lipid Metabolism and Endocrine Resistance in Prostate Cancer, and New Opportunities for Therapy. Int J Mol Sci. 2019 May 28;20(11):2626. PMID: 31142021.\u003c/li\u003e\n\u003cli\u003eCai M, Song XL, Li XA, et al. Current therapy and drug resistance in metastatic castration-resistant prostate cancer. Drug Resist Updat. 2023 May;68:100962.PMID: 37068396.\u003c/li\u003e\n\u003cli\u003eCui X, Wang S, Cao H, et al. A Review: The Bioactivities and Pharmacological Applications of Polygonatumsibiricum polysaccharides. Molecules. 2018 May 14;23(5):1170. PMID: 29757991. \u003c/li\u003e\n\u003cli\u003eZhang H, Li H, Pan B, et al. Integrated 16S rRNA Sequencing and Untargeted Metabolomics Analysis to Reveal the Protective Mechanisms of Polygonatum sibiricum Polysaccharide on Type 2 Diabetes Mellitus Model Rats.Curr Drug Metab. 2023;24(4):270-282. PMID: 37038712.\u003c/li\u003e\n\u003cli\u003eWang Y, Lan C, Liao X, et al. Polygonatum sibiricum polysaccharide potentially attenuates diabetic retinal injury in a diabetic rat model. J Diabetes Investig. 2019 Jul;10(4):915-924. PMID: 30426692.\u003c/li\u003e\n\u003cli\u003eYang JX, Wu S, Huang XL, et al. Hypolipidemic Activity and Anti-atherosclerotic Effect of Polysaccharide of Polygonatum sibiricum in Rabbit Model and Related Cellular Mechanisms. Evid Based Complement Alternat Med. 2015;2015:391065. PMID: 26089938.\u003c/li\u003e\n\u003cli\u003eYe G, Zhao Y, Zhu J, et al. Synergistic Effect of Polydatin and Polygonatum sibiricum Polysaccharides in Combating Atherosclerosis via Suppressing TLR4-Mediated NF- \u0026kappa; B Activation in ApoE-Deficient Mice. Evid Based Complement Alternat Med. PMID: 35845572.\u003c/li\u003e\n\u003cli\u003eDu L, Nong MN, Zhao JM, et al. Polygonatum sibiricum polysaccharide inhibits osteoporosis by promoting osteoblast formation and blocking osteoclastogenesis through Wnt/\u0026beta;-catenin signaling pathway. Sci Rep. 2016 Aug 24;6:32261. PMID: 27554324.\u003c/li\u003e\n\u003cli\u003eLong T, Liu Z, Shang J, et al.Polygonatum sibiricum polysaccharides play anti-cancer effect through TLR4-MAPK/NF-\u0026kappa;B signaling pathways. Int J Biol Macromol. 2018 May;111:813-821.PMID: 29343453.\u003c/li\u003e\n\u003cli\u003eXu Y, Guo Y, Lu C, et al.Polygonatum sibiricum polysaccharide Inhibited Liver Cancer in a Simulated Tumor Microenvironment by Eliminating TLR4/STAT3 Pathway. Biol Pharm Bull. 2023;46(9):1249-1259.PMID: 37661404.\u003c/li\u003e\n\u003cli\u003eHan SY, Hu MH，Qi GY，et al. Polysaccharides from Polygonatum Inhibit the Proliferation of Prostate Cancer-Associated Fibroblasts. Asian Pac J Cancer Prev. 2016;17(8):3829-33. PMID: 27644624.\u003c/li\u003e\n\u003cli\u003eLi M, Liu Y, Zhang H, et al. Anti-cancer Potential of Polysaccharide Extracted From Polygonatum sibiricum on HepG2 Cells via Cell Cycle Arrest and Apoptosis. Front Nutr. 2022 Jul 4;9:938290. PMID: 35903453.\u003c/li\u003e\n\u003cli\u003eLi L, Tian LN, Ren ZX, et al. Research progress on the structural analysis and functional activity of polysaccharides. Chin. J. Exp. Tradit. Med. Fromul. 2015, 21,231\u0026ndash;234. DOI:10.13422/j.cnki.syfjx.2015150231(in Chinese).\u003c/li\u003e\n\u003cli\u003eTewari D, Patni P, Bishayee A, et al. Natural products targeting the PI3K-Akt-mTOR signaling pathway in cancer: A novel therapeutic strategy. Semin Cancer Biol. 2022 May;80:1-17.PMID: 31866476.\u003c/li\u003e\n\u003cli\u003eHashemi M, Taheriazam A, Daneii P, et al. Targeting PI3K/Akt signaling in prostate cancer therapy. J Cell Commun Signal. 2023 Sep;17(3):423-443. PMID: 36367667.\u003c/li\u003e\n\u003cli\u003eWang Q, Wu S, Gu Y, et al.RASAL2 regulates the cell cycle and cyclin D1 expression through PI3K/AKT signalling in prostate tumorigenesis. Cell Death Discov. 2022 Jun 6;8(1):275. PMID: 35668070. \u003c/li\u003e\n\u003cli\u003eGu Y, Liang C. TRAIP suppressed apoptosis and cell cycle to promote prostate cancer proliferation via TRAF2-PI3K-AKT pathway activation. Int Urol Nephrol. 2023 Dec 15. PMID: 38100027.\u003c/li\u003e\n\u003cli\u003eRaja Singh P, Sugantha Priya E, Balakrishnan S, et al. Inhibition of cell survival and proliferation by nimbolide in human androgen-independent prostate cancer (PC-3) cells: involvement of the PI3K/Akt pathway. Mol Cell Biochem. 2017 Mar;427(1-2):69-79. PMID: 28025797.\u003c/li\u003e\n\u003cli\u003eChen H, Zhou L, Wu X, et al. The PI3K/AKT pathway in the pathogenesis of prostate cancer. Front Biosci (Landmark Ed). 2016 Jun 1;21(5):1084-91. PMID: 27100493.\u003c/li\u003e\n\u003cli\u003eLiu D, Tang W, Han C, et al. Advances in Polygonatum sibiricum polysaccharides: Extraction, purification, structure, biosynthesis, and bioactivity. Front Nutr. 2022 Dec 5;9:1074671. PMID: 36545471. \u003c/li\u003e\n\u003cli\u003eLiao YD, Grobholz R, Abel U, et al. Increase of AKT/PKB expression correlates with gleason pattern in human prostate cancer. Int J Cancer. 2003 Nov 20;107(4):676-80. PMID: 14520710.\u003c/li\u003e\n\u003cli\u003eMalik SN, Brattain M, Ghosh PM, Immunohistochemical demonstration of phospho-Akt in high Gleason grade prostate cancer. Clin Cancer Res. 2002 Apr;8(4):1168-71.PMID: 11948129.\u003c/li\u003e\n\u003cli\u003eLien EC, Dibble CC, Toker A. PI3K signaling in cancer: beyond AKT. CurrOpin Cell Biol. 2017 Apr;45:62-71. PMID: 28343126.\u003c/li\u003e\n\u003cli\u003eZhu S, Jiao W, Xu Y, et al. Palmitic acid inhibits prostate cancer cell proliferation and metastasis by suppressing the PI3K/Akt pathway. Life Sci. 2021 Dec 1;286:120046. PMID: 34653428.\u003c/li\u003e\n\u003cli\u003eZhang L, Shao L, Creighton CJ, et al. Function of phosphorylation of NF-kB p65 ser536 in prostate cancer oncogenesis. Oncotarget. 2015 Mar 20;6(8):6281-94. PMID: 25749044.\u003c/li\u003e\n\u003cli\u003eErdogan S, Serttas R, Turkekul K, et al. The synergistic anticancer effect of salinomycin combined with cabazitaxel in CD44\u003csup\u003e+\u003c/sup\u003e prostate cancer cells by downregulating wnt, NF-\u0026kappa;B and AKT signaling. Mol Biol Rep. 2022 Jun;49(6):4873-4884. PMID: 35705771.\u003c/li\u003e\n\u003cli\u003eDavoudi Z, Akbarzadeh A, Rahmatiyamchi M, et al. Molecular Target Therapy of AKT and NF-kB Signaling Pathways and Multidrug Resistance by Specific Cell Penetrating Inhibitor Peptides in HL-60 Cells. Asian Pac J Cancer Prev. 2014;15(10):4353-8. PMID: 24935396.\u003c/li\u003e\n\u003cli\u003eDuan C, Yu M, Xu J, et al. Overcoming Cancer Multi-drug Resistance (MDR): Reasons, mechanisms, nanotherapeutic solutions, and challenges. Biomed Pharmacother. 2023 Jun;162:114643. PMID: 37031496.\u003c/li\u003e\n\u003cli\u003eChen T, Xiao Z, Liu X, et al. Natural products for combating multidrug resistance in cancer. Pharmacol Res. 2024 Apr;202:107099. PMID: 38342327. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"clinical-and-experimental-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"clem","sideBox":"Learn more about [Clinical and Experimental Medicine](https://www.springer.com/journal/10238)","snPcode":"10238","submissionUrl":"https://submission.nature.com/new-submission/10238/3","title":"Clinical and Experimental Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"PSP, PC-3 cells, invasiveness, migratory ability, P65, PI3K and AKT","lastPublishedDoi":"10.21203/rs.3.rs-4668118/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4668118/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePolysaccharide extracted from polygonatum sibiricum (PSP) is a traditional herbal medicine component thought to have therapeutic effects on many diseases. In our study, we initially investigated the role of PSP in the development of human prostate cancer PC-3 cells and explored the potential mechanisms. First, the different concentrations of PSP were used to act on human prostate cancer cells PC-3, It was found that the proliferation, invasiveness, and migratory ability of PC-3 cells were significantly inhibited with increasing concentrations of PSP; the cell apoptosis rate and expression of caspase-3 increased with increasing concentrations of PSP; and the cell cycle was arrested in the S phase. In addition, we found that the expression of the MDR-1 gene and its encoding protein P-gp in PC-3 cells decreased after the treatment with PSP, so PSP might reverse multidrug resistance in PC-3 cells. Second, the possible mechanism of PSP action on PC-3 cells has been investigated, and the results showed that phosphorylated P65, PI3K, and AKT decreased in a concentration-dependent manner, as P65, PI3K, and AKT are molecules of the NF-kB and PI3K/Akt signaling pathways, so the potential mechanism of PSP on prostate cancer cells might be mediated by the PI3K/Akt and NF-kB signaling pathway simultaneously.\u003c/p\u003e","manuscriptTitle":"The potential antitumor activity of polysaccharide extracted from polygonatumsibiricum on human prostate cancer PC-3 cells ","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-29 13:03:33","doi":"10.21203/rs.3.rs-4668118/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorAssigned","content":"","date":"2024-07-04T09:36:57+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-03T12:59:28+00:00","index":"","fulltext":""},{"type":"submitted","content":"Clinical and Experimental Medicine","date":"2024-07-01T12:41:29+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"clinical-and-experimental-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"clem","sideBox":"Learn more about [Clinical and Experimental Medicine](https://www.springer.com/journal/10238)","snPcode":"10238","submissionUrl":"https://submission.nature.com/new-submission/10238/3","title":"Clinical and Experimental Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"8c5ea19d-655c-4522-9f44-f2ccada11aec","owner":[],"postedDate":"July 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-07-29T13:03:33+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-29 13:03:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4668118","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4668118","identity":"rs-4668118","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}