Inhibitory Effects And Mechanism Of Oleuropein And Isoorientin On Glioma

Background:Gliomas are the most common malignant primary brain tumors in adults. Despite significant advances in medical and surgical management during the past decades, prognosis of the disease remains poor, with a median survival of approximately 12-14 months. The poor prognosis and high lethality of the disease are primarily due to the fact that glioma cells are highly locally invasive and migratory, which leads to incomplete surgical resection and high frequency of tumor recurrence. In addition, glioma cells recruit nonmalignant cells to facilitate tumor progression. Macrophages are the predominant infiltrating immune cells recruited by glioma cells in glioma microenvironment. Recent studies indicated that macrophages infiltration correlates positively with glioma aggressiveness. The above-mentioned problems indicate that it is required to find novel therapeutic approaches that target both glioma cells and the glioma microenvironment.Tumor-associated macrophages are the most abundant immune cells resident in the tumor microenvironment, accounting for>10% of infiltrative cells in peri-tumoral GBM tissue. There are two main pathways for macrophage activation:the classical pathway (also referred to as M1) and the alternative pathway (also referred to as M2). The classical activated macrophages ((also called M1) are induced by IFN-y and Toll-like receptor (TLR) agonists such as LPS etc, express high levels of MHC class Ⅱ and CD86 and secrete large amounts of TNF-a, IL-6, and NO. Alternatively activated macrophages (also called M2) are induced by Th2-producing cytokines IL-4 and IL-13, express low levels of MHC class II and CD86 but release anti-inflammatory factors such as IL-10 and TGFβ. Cumulative research suggests that macrophages within malignant gliomas are dominantly the M2 subtype, the presence of M2 macrophages in glioma is correlated to poor prognosis.Several phytochemicals have been used for the treatment and prevention of glioma. Oleuropein, the main phenolic secoiridoid from the olive tree, has shown some promising anticancer activity in various types of cancer including breast cancer, thyroid cancer, colorectal cancerand prostate cancer. These investigations also suggest that key cellular signaling pathway such as NF-κB, PI3K-Akt, MAPK and HIF-1α are associated to the anticancer properties of oleuropein. Gulcin Tezcan has reported that Olea europaealeaf extract could inhibit the proliferation of glioma. Although oleuropein is one of the main components of Olea europaealeaf extract, effects and mechanisms of oleuropein on human glioma are still unknown.Isoorientin (3’,4’,5,7-tetrahydroxy-6-C-glucopyranosyl flavone; ISO), is a common C-glycosyl flavone in the human diet and is found in several plant species, such as Arum palaestinum, Phyllostachys pubescens, Lythrum salicaria, buckwheat, Patrinia villosa Juss, and Drosophyllum lusitanicum. Recent investigations show that ISO possesses several biological effects of pharmacologic importance, like anti-inflammatory, antioxidant, anti-bacterial and anti-nociceptive activities. Several studies have also shown that ISO have potent antitumor activity [13-15]. However, the effects of ISO on human gliomas cells and the mechanisms responsible for these effects have not been elucidated.Objective1. Inhibitory effects and mechanism of Oleuropein on glioma.(1) Investigate the effect of oleuropein on proliferation of glioma cells.(2) Examine the effect of oleuropein on macrophage migration amd M2 polarization.(3) Examine the effect of oleuropein on STAT3 pathway in glioma cells.(4) Examine the effect of oleuropein on the genes that aree regulated by STAT3 (IL-6、IL-8 and miR-21) in glioma cells.2. Inhibitory effects and mechanism of isoorientin on glioma.(1) Investigate the effects of isoorientin on proliferation of glioma cells.(2) Examine the effect of isoorientin on Wnt/beta-catenin pathway in glioma cells.(3) Examine the effect of isoorientin on miR-23a in glioma cells.(4) Examine the involvement of miR-23a in the regulatory mechanism of wnt/beta-catenin signaling pathway in glioma cells.Methods1. Inhibitory effects and mechanism of oleuropein on glioma.(1) In order to investigate the effect of oleuropein on proliferation of glioma cells, U87 and U251 glioma cells were the research object, MTT assay and BrdU labeling assay were used.(2) To examine the effect of oleuropein on macrophage infiltration into glioma stroma, THP-1 cells were the research object, and Transwell migration assay were used.(3) In order to examined the effect of oleuropein on the expression of M2 markers by tumor-associated macrophages, ELISA were used to examine the levels of cytokines IL-10 by macrophages, Western-Blot were used to examine the levels of phosphorylation of STAT3 and STAT3 in macrophages, and Immunoflourescence Assay were used to examine the levels of CD 163 by macrophages.(4) In order to examine the effect of oleuropein on STAT3 pathway and its downstream genes in glioma cells, U87 and U251 glioma cells were the research object, Western-Blot were used to examine the levels of phosphorylation of STAT3 and STAT3, ELISA were used to examine the levels of cytokines IL-6 and IL-8, RT-PCR were used to examine the levels of mir-21 and miR-181b.(5) Four week old male nude mice (BALB/c-nude) are used to examine the tumorigenicity. A total of 100 μL (containing 5×106 U251 cells) cell solutions were subcutaneously injected into the right flank of the mice. When the tumors had reached a mean size of 50-100 mm3, the mice were randomly divided into two groups (n=5). For oleuropein treatment, the animals were administered by oral gavage (500mg/kg/day) 5 days per week for 4 weeks. The tumor volume was measured once a week and the tumor volumes were calculated using the formula:V:=0.52x[LxW2], (V=volume, L=length, and W=width). The tumor tissue were removed after four weeks, immunohistochemistry assay were used to examine the number of CD 163 positive cells (M2 macrophages), Western-Blot were used to examine the levels of phosphorylation of STAT3 and STAT3, RT-PCR were used to examine the levels of miR-21 and miR-181b.2. Inhibitory effects and mechanism of isoorientin on glioma.(1) In order to investigate the effect of isoorientin on proliferation of glioma cells, U87 and U251 glioma cells were the research object, MTT assay and BrdU labeling assay were used.(2) In order to examine the effect of isoorientin on Wnt/beta-catenin pathway and miR-23a in glioma cells, U87 and U251 glioma cells were the research object, Western Blot was used to examine the levels of total and nucleus beta-catenin expression, RT-PCR were used to examine the levels of miR-23a.(3) In order to examine the involvement of miR-23a in the regulatory of wnt/beta-catenin pathway, gliomas were transfected with the miR-23a inhibitor or its negative control using the Lipofectamine 2000 reagent.(4) In order to investigate the effect of isoorientin in vivo, the subcutaneous xenograft model in nude mice was used. At last, the tumor tissue were removed, Western Blot was used to examine the levels of total and nucleus beta-catenin expression, RT-PCR were used to examine the levels of miR-23a.Results1. Inhibitory effects and mechanism of oleuropein on glioma.(1) MTT showed that:Oleuropein significantly suppressed the cell viability of U87 and U251 cells in a time-and dose-dependent manner. The BrdU labeling assay showed that oleuropein treatment strongly inhibited cell proliferation of glioma cells.(2) Transwell migration assay showed that:pretreatment with oleuropein inhibited the migration of THP-1 cells towards glioma cells.(3) Oleuropein decreased expression of M2 markers by tumor-associated macrophages.1) ELISA analysis showed that:Oleuropein treated glioma cells supernatant decreased the secretion of anti-inflammatory cytokines IL-10 by macrophages compared with the control supernatant.2) Western Blot analysis showed:Phospho-STAT3 was at a lower level in macrophages stimulated by oleuropein treated glioma cells supernatant. No significant changes were found about STAT3 level.3) Immunoflourescence Assay showed that:Supernatant of oleuropein treated glioma cells induced less CD163+ macrophages.(4) Oleuropein inhibited STAT3 pathway in glioma cells.1) Western Blot analysis showed that:Compared with control cells, both STAT3 and phosphorylation of STAT3 levels were reduced by oleuropein treatment.2) ELISA analysis showed that:Treatment with oleuropein attenuated IL-6 and IL-8 expression signifinantly in glioma cells.3) RT-PCR analysis showed that:Oleuropein treatment decreased the relative expression of miR-21 but not miR-181b in glioma cells.The results above indicated that Oleuropein inhibited STAT3 pathway and its downstream genes in glioma cells.(5) In vivo effects of oleuropein.1) Oleuropein treatment was found to significantly reduce the tumor growth in mice.2) Immunohistochemistry assay indicated that oleuropein treatment significantly reduced CD 163 positive M2 macrophage infiltration.3) RT-PCR analysis showed that oleuropein treatment decreased the relative expressions of miR-21 in vivo4) Western blotting analysis confirmed that oleuropein treatment reduced both STAT3 and phosphorylation of STAT3 levels in vivo.2. Inhibitory effects and mechanism of isoorientin on glioma.(1) MTT showed that:Isoorientin significantly suppressed the cell viability of U87 and U251 cells in a time-and dose-dependent manner. The BrdU labeling assay showed that isoorientin treatment strongly inhibited cell proliferation of glioma cells.(2) Western Blot analysis showed that:Compared with control cells, both total and nucleus beta-catenin expression levels were reduced by isoorientin treatment. RT-PCR analysis showed that:isoorientin treatment decreased the relative expression of miR-23a.(3) Western Blot analysis showed that:miR-23a inhibitor down regulated both total beta-catenin and nucleus beta-catenin expression in glioma cells.(4) In vivo effects of isoorientin.1) Isoorientin treatment was found to significantly reduce the tumor growth in mice.2) Western blot analysis confirmed that isoorientin treatment reduced both total and nucleus beta-catenin expression levels in vivo.3) RT-PCR analysis showed that:isoorientin treatment decreased the relative expression of miR-23a in vivo.ConclusionsIn summary, we have demonstrated for the first time that oleuropein inhibited glioma cell proliferation and glioma-mediated macrophage migration and M2 polarization through STAT3 signaling. Furthermore, our study has also shown that the downstream genes of STAT3 (IL-6, IL-8 and miR-21) are associated with the activity of oleuropein in glioma cells.In addition, we have demonstrated for the first time that isoorientin could inhibit glioma cell proliferation and identified Wnt/beta-catenin as targets for the activity of isoorientin. Furthermore, our study has shown for the first time that miR-23a was involved in the regulatory of wnt/beta-catenin pathway and isoorientin could regulate wnt/beta-catenin pathway through miR-23a in glioma cells.