| BackgroundMalignant glioma, originated from brain glial cells, is the most common type of adult intracranial primary tumors. It contains anaplastic astrocytoma, gliasarcoma, glioblastoma multiforme (GBM. Although these tumor accounts for only 2% of all adults tumors, they are still the fourth leading cause of death resulting from tumor, and it is a serious threat to human health. GBM, for example, the ratio between male and female is 1.6:1, and the average survival period was 14.6 months. At present, surgical removal with the adjuvant radiotherapy and chemotherapy is a major treatment for malignant glioma.Because of the high proliferation and strong invasion, malignant glioma is difficult to completely removed, even if exerted the adjuvant radiotherapy and chemotherapy after surgery. So patients has no significant long-term survival. Over the past few decades, new strategy and new method of glioma therapy have constantly appeared,such as gene therapy, immunotherapy, photochemical therapy, molecular targeted therapy, neural stem cells therapy. But few methods in clinic has notable success.Apoptosis and invasion are hot spots in the research of glioma.Many studies showed that the occurrence of glioma is the result of not only excessive preliferation but also huge accumulation, and inhibition of apoptosis can promote the development of glioma.Different pathways of apoptosis has been confirmed in glioma and proapoptotic factor, antiapoptotic factor and apoptosis related pathways all involved in the occurrence and development of glioma.Moreover, some apoptotic proteins are closely related to malignant degree and the the prognosis of patients. Among them, the casepase-3 is the core proteins of apoptotic process.The Bcl-2 family is the most important part in the study of glioma cell apoptosis, including Bcl-2 and Bax. Bcl-2 can inhibit cell apoptosis and Bax can promote cell apoptosis. The study found that the more Bcl-2 expression in glioma, the lower degree of glioman will be. Bax is just the opposite. P53 was the firstly found to be related to apoptosis of tumor suppressor genes.wild-type P53 can raise expression of Bax gene, which indirectly promote cell apoptosis. Some articles showed the expression of Survival has a relation with the degree of glioma. Apoptosis is closely related to the occurrence of glioma.At present some new purpose of glioma chemotherapy is to induce glioma cell apoptosis, rather than to inhibit their proliferation. Therefore.it is important to further study the mechanism of apoptosis,then use effective drug or find new targets to promote apoptosis of glioma.At the same time, the occurrence of glioma development is closely related with powerful invasion.Autopsy has reported that 45% of the growth of GBM range over a lobes,25% throughout the hemisphere,25-30% to the other hemisphere. Study found that gliomas mostly spread through the white matter and the extracellular matrix. The damage of extracellular matrix and cell surface adhesion molecules, which include far protein, protein fiber connection, laminin, integrin, hyaluronic acid, salt and other molecules, is the molecular basis of invasion. Matrix metalloproteinases family, especially MMP2 and MMP9, can degrade many kinds of extracellular matrix components, destruct of glioma cells invade histology barrier, and play a key role in the process of invasion and metastasis of glioma cells. So our work is to restrain activity of MMP2 and MMP9 and achieve the purpose of inhibition of glioma cells invasion.At present, there are no specifically clinical drugs to promote glioma cell apoptosis and control the invasion. The major chemotherapy drug istemozolomide, due to its cytotoxicity, with less adverse reaction, but the high price. So it is particularly important to look for better chemotherapy drugs. Now, with the deep research and development of traditional Chinese medicine, a lot of clinical trials confirmed the effect of Chinese medicine on treatment for glioma. Nitidine chloride is one form of Nitidine, and many studies have shown that it can suppress malignant biological behavior of osteosarcoma, stomach, liver and kidney cells. Some reports also show that AKT and ERK signal pathway mayparticipate in the anti-tumor process of NC. However, the effect of NC on glioma cells has not been reported. In this article we will study the potential function and related mechanisms of NC on glioma cells.Oleuropein, extracted from the olive tree, has shown the anti-cancer effect in many tumors, including thyroid cancer, breast cancer, colorectal cancer and prostate cancer. These studies also reveal that cellar signal pathways including MAPK, PI3K, are associated with the anti-tumor properties of oleuropein.However, fuctions and mechanisms of oleuropein in human gliomas are seldom reported. And we will deeply study the effects and underlying molecular mechanisms of oleuropein on gliomain the second part of this work.Objective1.NC inhibits the malignant biological behavior of glioma cells:(1) Study the effect of NC on proliferation ofglioma cell.(2) Study the effect of NC on migration and invasionglioma cell.(3) Examine mitochondrial membrane potential and mitochondrial morphology,generation of lactic acid and ATP.(4) Study the effectof NC on PI3K/AKT/mTOR pathway in glioma cells.2. Oleuropein inhibits the proliferation and invasion of glioma cells:(1) Study the effect of Oleuropein on proliferation ofglioma cell.(2) Study the effect ofOleuropein on migration and invasionglioma cell.(3) Study the effect ofOleuropein on glioma cell apoptosis.(4) Study the effectof Oleuropeinon AKT, P38, ERK and JNK pathway.(5) Study the effectof Oleuropeinon downstream molecules of AKT pathway (P53, Bax, Bcl-2, Cas-3, Cas-9, MMP-3 and MMP-9).Methods1.NC inhibits the malignant biological behavior of glioma cells:(1) U87 and U251 glioma cells were chosen to investigate the function of NC on proliferation of glioma cell. CCK-8 assay and EdU labeling assay were used.(2) With U87 and U251 glioma cells, scratches and Transwell assay are used to test impacts of NC on migration and invasion of glioma cells.(3) With U87 and U251 glioma cells. Measurement of mitochondrial membrane potential (Δψm), mitochondrial morphology, ATP and lactate to examine the anti-tumor effects of NC on glioma cells.(4) With U87 and U251 glioma cells, Western-Blot were used to examine the expression of AKT, p-AKT, mTOR and p-mTOR.(5) Four week old BALB/c nude mice as the research object, the 100μ L (contains 1 x 107 U87 cells) cell solutions were injected into the right side of the brain cortex and were randomly divided into two groups:NC-treated group and control group. For NC-treated group, the animals were administered by oral gavage (500 mg/kg/day) 5 times a week for 4 weeks and the control groups were given normal saline.Every 14 days animal tumor size was measured in vivo imaging system. Survival time and survival curve ofmices in two groups were drawn.2.Oleuropein inhibits the proliferation and invasion of glioma cells:(1) A172 and U251 glioma cells were chosen to investigate the function of Oleuropein on proliferation of glioma cell. CCK-8 assay were used.(2) With A172 and U251 glioma cells, scratches and Transwell assay are used to test impacts of oleuropein on migration and invasion of glioma cells.(3)With A172 and U251 glioma cells, flow cytometry technology is used to research the impact of oleuropein on glioma cell apoptosis.(4) With A172 and U251 glioma cells, Western Blot is used to examine the changes of AKT, P38, ERK and JNK pathway after use of oleuropein and the influence of oleuropein on the downstream molecules (P53, Bax, Bcl-2, Cas-3, Cas-9, MMP-3 and MMP-9).Results1.NC inhibits the malignant biological behavior of glioma cells:(1) The results of CCK-8 assay showed astable decrease in cell viability after treatment with NC, which indicated NC can inhibit the viability of glioma cells.(2) The results of scratch assay and Transwell assayindicated thatNC significantly restrained the migration and invasion of glioma cells.(3) NC inhibits production of ATP and L-lactate in GBM cells; mitochondrial activity was suppressed in the NC-treated U251and U87 cells relative to the untreated cells; NC-treated cellsfavored the fission state, while in the untreated GBM cells thefusion state predominated;(4) Treatment with NCled to increases in Bax and cytochrome c and simultaneousdecreases in Bcl-2 compared to the untreated U251 and U87cells; Increasing concentrations of NC suppressedphosphorylation of both Akt and mTOR, indicating thatPI3K/Akt/mTOR signaling was inhibited by NC.(5) NC-treatment can obviously reduce the growth of glioma cells. Survival of NC-treated groups was obviously prolonged.2. Oleuropein inhibits the proliferation and invasion of glioma cells:(1) The results of CCK-8 assay showed astable decrease in cell viability after treatment with oleuropein, which indicated Oleuropein can inhibit the viability of glioma cells.(2) The results of scratch assay and Transwell assayindicated thatoleuropein significantly restrained the migration and invasion of glioma cells.(3) The results of Flow cytometry revealed that oleuropein induced dose-dependent apoptosis in U251 and A172 cells.(4) Western-Blot technique has confirmed that oleuropein-treatment significantly inhibited the phosphorylation of AKT in glioma cells, but not phosphorylation of ERK、JNK、P38. Besides, the expression of MMP-2 and MMP-9 were significantly decreased afteroleuropein treatment. The expression of Bax was increased whilethe expression of Bcl-2 was decreased after treatment witholeuropein. Additionally, the expression of cleavedcaspase-9 and -3 were increased.Conclusions1.Vitro and vivo tests show that NC can inhibit the proliferation, migration and invasionofgliomacells, and obviously suppressactivity of PI3K/AKT/mTOR pathway, which could exert influence onexpression of Bax, Bcl-2, and Cyto-C.2.Oleuropein can inhibit the proliferation, migration and invasion of glioma cells viasuppression of AKT activation, and promote the apoptosisgliomacells. On the other hand, expressionsof MMP2 and MMP9 decrease, which effectively restrain the migration and invasion ofglioma cells. |
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