Anti-cancer Effects Of Fucoxanthin On Glioma Cells And Its Underlying Mechanism

BackgroundGliomas comprise primary central nervous (CNS) system tumors, which are derived fromneuroepithlial tissue.Gliomas are the most frequent tumors in CNS, accounting for 40%. Gliomas can be classcificed grade I to IV in term of a histological/prognostic grading system. Acoording to the grading system,glioblastoma multiforme (GBM) is the kind of Grade IV gliomas, which is the most malignancy gliomas. GBM grow uncontrolled, and invade into neighboring brain tiusses or structures.Traditional treatments such as surgical resection, radiotherapy and chemotherapy are difficult to achieve achieves effective at curing primary due to poor prognosis. Glioma is one that seriously effects human healthy.Throughout the past 20 years, molecular biology achevied great progress on biological characteristics and molecular mechanism of GBM, that revealing the survival, apoptosis, autophagy, migration of GBM and supplying new therapeutic targets for curing GBM. But molecular regulation is difficult and complicated to entirely understand. So it is necessary and meaningfur for us to work on it, and it still the hot area of GBM at present.The extracted and purification technique of the Chemical Component is making progress. The extracts of medicinal herbs provide a novel remission or cure way to gliomas or other cancers. Fucoxanthin is a major carotenoid extraction from seaweedsand its molecule formulacomprises an unusual allenic bond andconjugated carbonyl group.Because of these special functional groups, fucoxanthin represents antioxidant activity, induction of apoptosisin cancer cells.Additionally, current researches focus on the effect of fucoxanthin on inhibition of migration and invasion. It has been demonstrated that fucoxanthin reduces the ability of invasion and metastasis of melanoma cells and osteosarcoma cellsin vitro and in vivo. Unfortunately, few reports have been claimed the effect of fucoxanthin on glioma cells, particularly in the category of underlying mechanisms of glioma growth inhibition.There had been reported that PI3K/Akt/mTOR and MAPK signal pathways are significant to regulate the development and progression of tumor cells.The two signal patyway perform excessive phosphorylation in many malignant tumor including glioblastoma multiforme, which activate downstream gene, enhance malignant proliferation, anti-apoptosis and invasion. By down-regulating the excessive phosphorylation can achieve our aim to inhibit the proliferation, pro-apoptosis and anti-invasion.Purpose1. To investigate the effects of Fucoxanthin on inhibiting proliferation in U87 cells and normal neurons.2. To investigate the effects of Fucoxanthin on inducing apoptosis in U87 cells.3. To investigate the molecular mechanism of Fucoxanthin on inhibiting proliferation and inducing apoptosis in U87 cells4. To investigate the effects of Fucoxanthin on cell migration and invasion in U87 cells5. To investigate the molecular mechanism of Fucoxanthinon cell migration and invasionin U87 cells6. To investigate the effects of Fucoxanthin on inhibiting proliferation and inducing apoptosisin vivoMethods1. Investigating the effects of Fucoxanthin on inhibiting proliferation in U87 cells and normal neuronsCells were treated with Fucoxanthin (6.25,12.5,25,50,75,100μM) for 12,24, 48 h. Cell viability was analysised by MTT assay. Normal neurons were treated with Fucoxanthin (25,50μM) for 24 h. Cell viability was analysised by MTT assay.2. Investigate the effects of Fucoxanthin on inducing apoptosis in U87 cells.Cells were treated with Fucoxanthin(0,25,50μM) for 24 h and stained with DNA-specific fluorescent dye Hoechst 33342.The stained cells were visualized under a fluorescence microscope.The degree of nuclear condensation was evaluated and the apoptotic cells were quantified.Cells were treated with Fucoxanthin(0,25,50μM) for 24 h and stained with DIOC6(3). Mitochondrial membrane potential (△ψm) was analyzed by flow cytometer.Cells were treated with Fucoxanthin(0,25,50μM) for 24 h and stained with Annexin V-FITC/SYTOX Green. The stained cells were analyzed with flow cytometry.Cells were treated with Fucoxanthin(0,50μM) for 24 h. Cells were examined with Jeol 100 CX II transmission electron microscope at 60 kV after collecting and disposing.3. Investigating the effects of Fucoxanthin on apoptotic protein in U87 cellsCells were treated with Fucoxanthin(0,25,50μM) for 24 h. Immunoblot analysis was done forH2AX、Bcl-2、PARP、BAX、caspase-9、caspase-3.4. Investigating the molecular mechanism of Fucoxanthin on inducing apoptosis in U87 cellsU87 cells were treated with Fucoxanthin (0,25,50μM) for 24 h. Protein was extracted from each group were immunoblotted forAkt、p-Akt、mTOR、p-mTOR.To further prove that Fucoxanthin induced apoptosis by PI3K/Akt/mTOR signal pathway. Cells were treated with Fucoxanthin (0,50μM) and PI3K inhibitor LY294002 for 24 h. Cell extracts from each group were immunoblotted forAkt、p-Akt、 mTOR、p-mTOR、Bcl-2、BAX、Cleaved-capase-9.5. Scratch wound healing assayThe U87 cells were in 6-well platesand incubated for 12 h. The cell monolayer was scraped in a straight line using a 200-μL pipette tip and the cells were washed three times with PBS. The cells were incubated in free mediaand various concentrations of fucoxanthin (0,25,50μM) for 18 h.Images were captured under phase-contrast microscope.6. Trans-well migration assayU87 cells were resuspended with medium containing 1% FBS and seeded on trans-well chambers. Medium contained 20% FBS in the lower chambers served as the chemoattractant. Fucoxanthin (0,25,50μM) was added in the lower chambers. Invading cells were dyed with 0.1% crystal violet and captured by using Image-Pro Plus software under a phase-contrast microscope (magnification, x200). Crystal violet in the invading cells was dissolved with 33% acetic acid. The absorbance of crystal violet dissolved in acetic acid was measured at 570 nm using a microplate reader.7. Trans-well invasion assayTrans-well chambers were precoated withMatrige1.U87 cells were resuspendedwith medium containing 1% FBS and seeded on trans-well chambers. Medium contained 20% FBS in the lower chambers served as the chemoattractant. The indicated concentrations (0,25,50μM) was added in the lower chambers. Invading cells were dyed with 0.1% crystal violet and captured by using Image-Pro Plus software under a phase-contrast microscope (magnification, x200). Crystal violet was dissolved with 33% acetic acid. The absorbance of crystal violet dissolved in acetic acid was measured at 570 nm using a microplate reader.8. Investigating the effects of Fucoxanthin on invasive protein in U87 cellsDifferent concentration Fucoxanthin treated U87 cells with 24 h. Cell protein was extracted and immunoblotted by MMP-9, MMP-2, uPA.9. Investigating the molecular mechanism of Fucoxanthin on inhibiting invasive in U87 cellsU87 cells were treated with Fucoxanthin (0,25,50μM) for 24 h. Immunoblot analysis was used forp38, p-p38, ERK, p-ERKofdifferent group.P38 inhibitor SB203580 was added to further prove that Fucoxanthin induced apoptosis by p38 signal pathway. Cells were treated with Fucoxanthin (0,50μM) and p38 inhibitor SB203580 for 24 h. Cell protein from each group was immunoblotted forp38, p-p38, MMP-9, MMP-2.10. Investigating the effects of Fucoxanthin on inhibiting proliferation and inducing apoptosis in vivoU87 cell (5×106cells) was injected subcutaneously into the abdominal of nude mice. Treatment of mouse with fucoxanthin was initiated on the day after cell injection. Fucoxanthin was dissolved in soybean oil and 1 g/kg by oral gavage every day for 28 days. The size of the primary tumor was measured weekly. On day 28, all mice were euthanized. Primary tumors were dissected out for measurement of weight and staining with hematoxylin and eosin (H&E) and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) using a commercial kit.11. Statistical analysisAll measurements were performed in triplicate and all values are expressed as the mean±the standard error. The results were subjected to an analysis of variance (ANOVA) using Tukey’s test to analyze differences between means. In each case, a P value< 0.05 was considered statistically significant.Results1. Fucoxanthin inhibited cell proliferation and induced apoptosis in U87 cellsThe effect of fucoxathin on inhibition of U87 cell growth was detected by MTT assay, which was conducted in a concentration-and time-dependent manner. The MTT data revealed that treatment U87 cells with 25,50,75 and 100μM fucoxanthin significantly decreased the level of cell viability incubation for 12,24 and 48 h(P<0.05), compared with blank control and control groups. We can see that Fucoxanthin can inhibit U87 cells proliferation. The effect of fucoxathin on neurons cells viability. Compared with blank control, there were no significantly differences.To evaluate the toxic effect of U87 cells on apoptosis induced by fucoxanthin, the cellular nuclei were stained with hoechst 33342 and then assessed by microscopy. Observation with microscopy, fracted nuclei were dose-dependent in different groups (F=57.506,P<0.001),compared between groups indicatedthe inhibition effects are dose dependent (P<0.05). Similarly, the expression of H2AX, a hallmark of chromosome damage, was increased which indicated that fucoxanthin impaired U87 cells via apoptosis initiation. In addition, depolarization of mitochondrial potential (Δμm) is indispensable part involved in apoptotic program and we examined whether fucoxanthin caused apoptosis via disruption of mitochondria normal function. DiOC6(3) is a dye that suggests Δψm change by shifting its fluorescence emission from blue to yellow. The maximum absorption wavelength was blue shift after treatment U87 cells with different concentration of fucoxanthin. The disruption of Δψm and destroyed the mitochondrial functionwere dose-dependent in different groups (F=431.583, P<0.001),compared between groups indicatedthe disruption effects are dose dependent (P<0.05). Impaired mitochondrial membrane releases the cytochrome c into cytoplasm which activates the apoptosis progression. Apoptosis was detected in the fucoxanthin-incubated cells by performing FITC-Annexin/SYTOX Green double staining which could distinguish apoptotic cells. Apoptotic cells increased were dose-dependented (F=259.15, P<0.001).25μM and 50μMwere significant difference compared with that of control groups (P<0.05).Transmission electron microscope (TEM) could find some changes of ultrastructure, including organelle and chromosome. We visualized the phenomenon of early and late apoptosis after treatment of U87 cells with fucoxanthin by means of electron microscope.2. Fucoxanthin inducesapoptotic protein in U87 cellsWe examine the changes of protein expression between Bcl-2 and Bax, which are anti-apoptotic protein and pro-apoptotic protein. Fucoxanthin showed an increment in Bax expression and a decrement in Bcl-2 expression compared with the control cells. The balance of Bcl-2/BAX dominates the switch which turns on or off the apoptosis. Notably, fucoxanthin initiated the activation of apoptotic pathway via Bax folded expression and it transduces the signal to the downstream-caspase system [25]. As expected, the enhanced activation of cleaved-PARP, caspase-9 and -3 was showed in fucoxanthin-treated groups.3. Fucoxanthin induces apoptosis by PI3K/Akt/mTOR signal pathwayWe confirmed that whether the effect of fucoxanthin on apoptosis was caused by negative regulation of PI3K/Akt/mTOR pathway. We observed that the expression of phosphorylation of Akt was dramatically decreased by treatment of U87 cells with fucoxanthin at the concentration of 25 and 50μM, and the level of phosphorylated mTOR was found similar result only at higher concentration of fucoxanthin. These results suggested that fuocoxanthin indeed inhibited the PI3K/Akt/mTOR pathway by reduction of their phosphorylated forms. To further prove the positive effect of fucoxanthin on apoptosis via suppression of PI3K/Akt/mTOR system.We incubated U87 cells with PI3K inhibitor prior to fucoxanthin treatment (50 μM). Undoubtedly, the expressions of active form of Akt/mTOR were lessened by exposure of glioma cells with only fucoxanthin and PI3K inhibitor, individually. Ulteriorly, the application of LY294002 had the semblable bands with fucoxanthin of western blotting on the expression of Bax, Bcl-2, cleaved-caspase-9, which indicated that the inhibition of PI3K/Akt/mTOR exactly induced the activation of apoptosis. Eventually, the combination of LY294002 and fucoxathin had more effect on apoptosis related proteins, compared to that of fucoxanthin or PI3K inhibitor. These results demonstrated that the inductive effect of fucoxanthin on apoptosis is performed via suppression of PI3K/Akt/mTOR system.4. Fucoxanthin inhibits the invasion and migration of U87 cellsInvasiveness and migration are the pathological signatures of human malignant gliomas, the effects of fucoxanthin on the invasiveness and migration of U87 cells were checked by scratch wound healing assay and trans-well assays. The healing rate was decreasing with dose-dependent in different groups (F=145.252, P<0.001). The both lower and higher concentration of fucoxanthin exposure remarkably restrained the invasion of U87 cells showing by the reduced healing rate compared to that of untreated-cells (P<0.05). Another outcome of the in vitro, trans-well assay, certified that treatment of U87 cells with fucoxanthin apparently attenuated the invasion and migrating rate which certified that fucoxanthin indeed had ability to control the translocation of U87 cells to adjacent tissue or other parts of body. Besides, we further investigated the mechanisms underlying fucoxanthin-inhibited properties of migration and invasion of U87 cells. It has been proven that the increased degree of matrix metalloproteases (MMPs) expression is closely associated with invasiveness and migration of human gliomas. MMPs promote tumor growth, angiogenesis, metastasis and invasion by degradation of extracellular matrix to break down structure of tissue and cell or provide various growth factors. Therefore, we selected two kind of gelatinase, MMP-2 and MMP-9, as the objective to explore the potential mechanism of fucoxanthin on U87 cells. As expected, the protein levels of MMP-2 and MMP-9 were undoubtedly suppressed by incubation glioma cells with fucoxanthin at the concentration of 25 and 50μM. The serine protease urokinase-type plasminogen activator (uPA) is upstream signaling and regulates the expression of MMPs to influence the characteristics of malignant glioma cells. We observed that the expression of uPA was reduced by exposure of U87 cells with fucoxanthin suggesting the effect of drug on U87 cells via inhibition of uPA-MMPs systems. These findings indicate that the application of fucoxanthin inhibits the invasive and migrative ability of glioma cells in vitro.5. Fucoxanthin suppresses the capability of invasion and migration by inhibition of p38 signaling pathwayMMPs-caused invasion and migration of tumor cells are mediated by the family of mitogen-activated protein kinases (MAPKs). We preformed the western blotting to examine the expression of two isoforms of MAPK:ERK and p38. The degree of phosphorylated-p38 expression was markedly decreased in a concentration-dependent manner. Conversely, fucoxanthin abnormally increased the protein levels of phosphorylated-ERK which should promote the cancerous cells to invade or infiltrate into other parts. Therefore, to verified that if the p38 was the upstream target of MMPs which was inhibited by chemical in our article. As expected, the MMPs was significantly reduced by the p38 inhibitor (SB203580) treatment which indicated that the p38-MMPs signaling pathway involved the fucoxanthin-mediated the reduction of disruption of other normal tissue of U87 cells. Hence, fucoxanthin negatively regulated the activity of glioma cells on invasion and migration mediated by suppression of activation of p38.6. Fucoxanthin inhibits the growth of tumorigenicity of U87 cells in BALB/c-nude miceWe had demonstrated that fucoxanthin was capable of inhibition of proliferation, invasion and migration, and induction of apoptosis on U87 cells in vitro. Next, we inspected that whether fucoxanthin had the similar results in vivo. We assessed the anti-cancer capability of fucoxanthin using immune deficient nude mice (BALB/c-nude) as the testing model. In the first two weeks, we had seen significant diffrence between two groups. The volumes of tumor tissues of positive drug group were significant smaller than control groups’in the third week, and in the fourth week the difference became more obvious (P<0.05). We removed the tumors and weighed them. Compared with control groups, the disposed groups were much lighter (P<0.05). Additionally, we examined the function of fucoxanthin on U87 cells in vivo by histochemistry and immunohistochemistry methods. H&E staining illustrated that the cell density was declined and the morphology was changed with irregular cell shape, and scattered nuclei showing by light and uneven purple color. TUNEL assay certified that apoptosis was appeared after treatment U87 cell with fucoxanthin showing by staining aberrated chromosome with brown color.These results denote that administration of these mice with 1 g/kg/day of fucoxanthin after inoculation of U87 show significant reduction of the tumor volume and weight due to the emerging apoptotic cells in the U87 cells as analyzed by H&E staining and TUNEL assay. And then, we explored the underlying signaling pathway mediated by fucoxanthin in vivo via extraction proteins from tumor mass of U87 cells. As illustrated in Fig.6c, fucoxanthin enhanced the expression level of apoptotic-related proteins and inhibited the activation of PI3K/Akt/mTOR and p38 system to accomplish reduction of invasion and migration of U87 tumor. These results denote that administration of these mice with 200 mg/kg/day of fucoxanthin after inoculation of U87 cells significantly reduce of the tumor volume and weight due to the emerging apoptotic cells and decline of invasion and migration in the U87 cells.