Platycodin D Inhibits Prostate Cancer Tumor Growth And FOXO3a-related Mechanism In Vitro And In Vivo

Background:Prostate cancer is the second leading cause of cancer death in men. American CancerSociety (ACS) has reported28,170men died of prostate cancer in the United States in2012,and29,720in2013. The mortality rate in Asian countries is much lower but it is graduallyincreasing. Patients with advanced prostate cancer are generally treated by endocrine therapy.The symptom relief provided by endocrine therapy is temporary. In addition, most patientsunder the treatment exhibit progressive hormone-refractory prostate cancer (HRPC) after aperiod of remission, and suffer bone metastasis which induces pain and pathological fractures.Chemotherapy was established to treat patients who no longer respond to hormone therapy,however the side effects of chemotherapy must be considered, and some advanced prostatecancers are resistant to chemotherapy. Therefore, there is an urgent need to discover new drugsto treat prostate cancer, especially advanced disease.Natural products (NPs), semi-synthetic NPs and NP-derived compounds have beenapproved to be an important source of therapeutic agents, especially in anti-cancer drugdiscovery and development. Among the chemotherapeutic agents originated from naturalproducts, Saponins have demonstrated their inhibitory activities against human cancer cell linesand can be retrieved from various herbs.As one of the major triterpene saponin found in the root of Platycodon Grandiflorum,Platycodin D (PD, MW:1224.38; Fig.2) is commonly known as Jiegeng in China. It waspreviously reported that PD could induce apoptosis in gastric and breast cancer cells in vitro. Itsanti-cancer activities on prostate cancer and its molecular mechanisms of action are not wellunderstood. In this study, we explored the anti-tumor activities of PD against prostate cancermodels, and attempted to further elucidate the possible mechanism(s) of action of PD, in orderto provide a basis for the future development of this agent for human prostate cancer chemotherapy.FOXO3a, a tumor suppressor, likely functions as a trigger for apoptosis through itsupregulation of genes necessary for cell death. Deregulation of FOXO3a is associated withtumorigenesis. FOXO-mediated regulation of the CIP/KIP family has been reported. Bothp21WAF1/CIP1and p27KIP1play crucial roles in controlling cell proliferation. FOXO3a can inducecell cycle arrest by upregulating these cell cycle inhibitors (p21WAF1/CIP1and p27KIP1) andconsequently attenuating the activity of cell cycle-promoting CDKs. The rescue ofFOXO3a-induced cell cycle arrest by the overexpression of cyclin D may result from a titrationeffect upon the CKIs upregulated by FOXO3a, rather than rescuing the transcriptionaldownregulation of cyclin D. It has also been suggested that FOXO3a can induce apoptosis,because FOXO3a has emerged as part of an important effector arm of PI3K/Akt signaling thatdrivesthe expression of multiple genes.MDM2(Murine double minute2), an E3ubiquitin ligase, has been demonstrated to playimportant roles in human cancer therapy. A previous study revealed that MDM2induced thedegradation of p21, a universal cell cycle inhibitor that binds to cyclin-CDK complexes.Additionally, it was reported that the overexpression of p21results in the induction of Bax andpromotes apoptosis. Therefore, the observed downregulation of MDM2could have beenresponsible for the decreased cell cycle progression and increased apoptosis of prostate cancercells.It is known that FOXO3a is downregulated by the MDM2-mediated ubiquitin-proteasome pathway. In order to determine whether PD can still increase the expression ofFOXO3a in the presence of high levels of MDM2(because MDM2overexpression iscommon in human cancers), we performed a transfection assay. The results showed that PDcould increase the protein level of FOXO3a regardless of the overexpression of a plasmidencoding MDM2. Further work is required to fully characterize the relationship betweenthese two proteins with regard to their regulation by PD.Results:PD exerted cytotoxicity against three prostate cancer cell lines, PC3cell3, DU145cells,LNCaP cells, with half-maximal inhibitory concentrations in the range of11.16to26.13μmol/L.The PC3cells were the most sensitive to PD, whereas RWPE-1cells, the normal prostate cells,were not sensitive to PD. A further study in these cell lines showed that PD could potently affect cell proliferation (indicated by the bromodeoxyuridine assay), induce cell apoptosis(determined by Annexin V-FITC flow cytometry) and cause cell cycle arrest (indicated by PIstaining). After being treated with PD for48hours, the DU145and LNCaP cells were arrestedin the G0/G1phase, and the PC3cells were arrested in the G2/M phase. A Western blotanalysis indicated that PD decreased the expression of MDM2and increased the expressionlevels of FOXO3a, p21and p27in all three cell lines. In PC3cells, PD exposure decreased thelevels of cell cycle-related proteins, including p-FOXO3a, E2F1, CDK2, CDK4, CDK6,cyclinD1, Cdc2, CDK1, and cyclinB1. PD exposure increased the expression ofapoptosis-related proteins, including Bax, Cleaved Caspase8, Cleaved Caspase3and Caspase3,whereas it down-regulated the Bcl2, PARP, Caspase8and Caspase9protein expression.Real-time PCR experiments indicated that the same changes occurred at the transcriptionallevel for all of these targets. Moreover, PD dose-dependently inhibited the growth of PC3xenograft tumors in BALB/c nude mice. A Western blot analysis of the xenograft tumorsindicated that similar changes in protein expression also occurred in vivo.Conclusion:PD exhibits significant inhibitory activities against prostate cancer cells. MDM2and theFOXO3a transcription factor appear to be involved in the action of PD, which provides a basisfor future development of human prostate cancer therapy.