As such, the anti-proliferative activity of ICA II may be attributable to its ability to induce cell cycle arrest

As such, the anti-proliferative activity of ICA II may be attributable to its ability to induce cell cycle arrest. Metastatic progression is usually a complex, multi-step process wherein tumor cells undergo changes in their migratory, invasive, proliferative, phenotypic, and angiogenic properties that enable CGP 57380 them to expand and spread to distant metastatic sites within affected individuals.21C23 Both invasion and migration are key drivers of this metastatic process.12 A number of studies have explored the ability of ICA II to modulate the invasion and migration of lung, gastric, and esophageal cancer cells.10,16 Herein, we decided that ICA II was able to significantly inhibit DU145 PC cell invasion and migration. Autophagy serves as a catabolic process in eukaryotic cells and is a vital means of maintaining intracellular homeostasis in physiological and pathological contexts.6,24,25 While it can promote cell survival in some cases, in other settings autophagy can induce apoptotic cell death depending on the intracellular signaling pathways that are engaged in a given cell.6 Autophagic cell death is an alternative form of programmed cell death that is distinct from apoptosis and that has been observed in the context of PC.6,26 Autophagy is associated with a disruption of apoptotic induction, whereas the caspase activity that is induced during apoptosis can, in turn, disrupt autophagic processes. assessed autophagy via laser confocal fluorescence microscopy. Western blotting was further utilized to measure LC3-II/I, Beclin-1, P70S6K, PI3K, AKT, mTOR, phospho-AKT, phospho-mTOR, and phospho-P70S6K levels, with qRT-PCR being used to evaluate the expression of specific genes at the mRNA level. Results We found that ICA II was capable of mediating the dose- and time-dependent suppression of DU145 cell proliferation, causing these cells to enter a state of cell cycle arrest and apoptosis. We further decided that ICA II treatment was associated with significant impairment of prostate malignancy cell migration and invasion, whereas autophagy was enhanced in treated cells relative to untreated controls. Conclusion Our results indicate that ICA II treatment is usually capable of suppressing human prostate tumor cell proliferation and migration while enhancing autophagy via modulating the PI3K-AKT-mTOR signaling pathway. As such, ICA II may be an ideal candidate drug for the treatment of prostate malignancy. Keywords: icariside II, prostate malignancy, PI3K-AKT-mTOR, autophagy, apoptosis Introduction Prostate malignancy (PC) remains one of the leading causes of cancer and death among men.1 Radical prostatectomy is the main method used to treat localized prostate malignancy,2 while androgen deprivation therapy (ADT) is the most important treatment in patients with advanced-staged PC.3 While initially efficacious in those with androgen-sensitive PC, most patients eventually exhibit ADT resistance such that their disease is reclassified as castration-resistant PC (CRPC) and has a poor prognosis.2,3 As such, it is vital that novel treatments for CRPC be identified. Many natural products from traditional medicinal herbs have been leveraged to treat cancer in recent years. The flavanol glycoside icariside II (ICA II) is usually a primary compound isolated from the traditional Chinese medicinal compound Herba epimedii.4,5 ICA II has been found to exhibit a diverse array of biological and pharmacological activities, serving to fight cancer, sexual dysfunction, and osteoporosis in multiple studies.4,5 ICA II can inhibit the COX-2/PGE 2 pathway and induce mitochondria-dependent apoptosis in PC cells.6 ICA II is further reported to exhibit anticancer activity against many human cancer cell lines in vitro and in vivo, with such activity being related to the ability of this compound to impact apoptosis and cell cycle progression, as well as the JAK2-STAT3, MAPK-ERK, and -Catenin signaling pathways.6 Autophagy is a key catabolic process in eukaryotic cells.7 The role of autophagy in CGP 57380 cancer is complex. Several studies have reported that autophagy can both suppress tumor growth by inhibiting the accumulation of damaged organelles and misfolded protein aggregates, while also promoting the survival and consequent growth of established tumors.8,9 Recently, autophagy has been highlighted as a potentially viable therapeutic target for the treatments of CRPC.3,7 The phosphatidylinositol 3-kinase-protein kinase B-mammalian target of rapamycin (PI3K-AKT-mTOR) signaling pathway is an essential regulator of activities such CGP 57380 as cellular motility, proliferation, and autophagy.8C10 The present study was therefore designed with the goal of evaluating the impact of ICA II on human PC cell proliferation, migration, and autophagy and the mechanisms underlying such activity. Materials and Methods Materials Dulbeccos Modified Eagle Medium CGP 57380 (DMEM), fetal bovine serum (FBS), and penicillin/streptomycin were obtained from Gibco (Life Technologies, NY, USA). Phosphate buffered saline (PBS), protease and phosphatase inhibitor cocktails, bovine serum albumin (BSA), Radio-Immunoprecipitation Assay (RIPA) lysis buffer, stripping buffer, propidium iodide (PI), and thioglycollate were from Sigma Aldrich (St. Louis, MO, USA). An annexin V-FITC-base apoptosis detection kit, a Cell Counting Kit-8 (CCK-8), and Transwell chambers (with Matrigel pre-coating) were from BD Biosciences (San Jose, CA, USA). Antibodies specific for microtubule-associated protein 1A/1B-light chain 3 (LC3), Beclin1, P70S6K, PI3K, AKT, mTOR, phospho-AKT, phospho-mTOR, and phospho-P70S6K were from Cell Signaling (Santa Cruz, CA, USA). Ethics Statement DU145 cells were obtained from the Cell Lender of Chinese Academy of Sciences (Shanghai, China). All experimental procedures were carried out in accordance with the guidelines of the Chinese Care and Use legislation, and were approved by the Animal Ethics Committee of Beijing Tongren ZNF384 Hospital, Capital Medical University or college. Cell Culture DU145 cells were cultured in DMEM made up of 10% FBS and penicillin/streptomycin at 37C in a 5% CO2 incubator. Cell Proliferation Assay A CCK-8 assay was used to assess the impact of ICA II on DU145 cell proliferative activity. Briefly, DU145 cells were added to a 96-well plate and were treated for 12, 24, or 48 h using 0, 10, 20, 40, or 80 M ICA II. A CCK-8 kit was then used based on provided directions, with absorbance (OD) at 450 nm being evaluated via Multiclan Ex lover plate reader (Thermo Fisher.