Effects Of G-CSF And Melatonin On Proliferation,Migration And Invasion Of Glioma Cells

Glioma, which takes about50%of all primary tumors in adult brain, is the most common primary brain tumor. Glioma grows in an infiltrating model, which makes it extremely malignancy, with an average median survival less than1year. Recent years, although great advances have been achieved in glioma therapy, including surgery, radio-and chemotherapy, immunotherapy as well as gene therapy, glioma remains not to be cured. High proliferation, migration and invasion are the main characteristics that responding for the incurable of glioma. Therefore, the control of metastasis and invasion represents an important therapeutic target in the treatment of glioma. However, the present therapy strategies are limited in view of neural toxicity, hematologic toxicity, or difficulty in crossing the blood-brain barrier (BBB) of many drugs. Thus, development of new therapeutic strategies that are effective in inhibition of tumor progression and less toxicity as well as readily crossing the BBB is desirable. Also, new strategies are urgently needed with insight into the mechanisms that are involved in the proliferation and invasion of glioma cells.Granulocyte colony-stimulating factor (G-CSF) and melatonin, two medicines commonly used in clinic, both have the neuroprotective effect and easily in crossing the BBB. G-CSF is used in the treatment of granulocytopenia due to its function of promoting hemaetopoisis. G-CSF has been an important adjunctive therpy in cancer treatment, however, its effects on cancer cells remains to be fully investigated. It is reported that melatonin has anti-tumor effects, which makes it becoming an adjunctive therpy in glioma treatment.G-CSF is a member of growth factors family. G-CSF acts through binding to a specific receptor (G-CSFR). G-CSFR is expressed by some stem cells, which can differentiate to mature granulocytes following stimulation of G-CSF, in bone marrow. Outside the hematopoietic system, G-CSFR is expressed in neuron, and under ischemic, G-CSF signaling protects neuron against ischemic induced apoptosis. Furthermore, G-CSF signaling promotes proliferation and differentiation of neural stem cells (NSCs). Recently, G-CSF signaling is found associated with tumor genesis and progression. Persistent activation of G-CSF signaling induces tumor formation in hematopoietic system. G-CSF and G-CSFR are over-expressed in many tumors, such as melanoma, non small cell lung cancer, bladder cancer, prostate cancer as well as brain cancers. G-CSF promotes proliferation and invasion in many cancers; however, the role it plays in glioma is unclear.Melatonin (N-acetyl-5-methoxytryptamine) is an indolamine that is synthesized by the pineal gland and the suprachiasmatic nucleus as well as peripheral tissues. Melatonin exerts a neuroprotective effect in many pathological conditions of the central nervous system, including Parkinson's disease (PD), Alzheimer's disease (AD), and ischemic brain injury. Also, melatonin displays antioxidant effects and acts as a free radical scavenger. Recent interest on melatonin has been focused on its potential antitumor effects. Indeed, the antitumor effects of melatonin have been tested in many types of tumors, including prostate cancer, sarcomas, colorectal cancer, hepatocarcinomas, melanoma, ovarian cancer, breast cancer, as well as brain tumors. Melatonin inhibits cell proliferation and induces apoptosis in most cell lines of the aforementioned tumors and decreases tumor growth in murine tumor models. Furthermore, recent studies have reported that melatonin can inhibit tumor invasion through increased adhesion by elevating E-cadherin and β1-integrin expression or modulating microfilament, and decrease matrix metalloproteinase (MMPs) production. Anti-invasion effect of melatonin has been shown in human mammary epithelial cancer MCF-7cells. In glioma cells, millimolar concentration of melatonin can reduce growth and inhibit cell progression from G1to S phase of the cell cycle. However, the effect of melatonin on migration and invasion of glioma has remained to be explored and elucidated. This paper includes two parts, and we will discuss the effects of G-CSF and melatonin on proliferation, migration and invasion of glioma cells. Our results may be beneficial to target therapy of glioma.Part Ⅰ:Effects of G-CSF on proliferation, migration and invasion of glioma cells.1. Expression of G-CSF and G-CSFR in human gliomas and the correlation between expression levels and glioma gradesTo detect the expression of G-CSF and G-CSR in human gliomas,29glioma samples were collected. All the tumor samples were corroborated in final pathology and classified according to the2007World Health Organization (WHO) classification of tumors of the central nervous system. There were8WHO grade Ⅰ/Ⅱ glioma,9Ⅲ anaplastic glioma and12Ⅳ glioblastoma, as well as5normal brain tissues. Total RNA and protein were obtained and expression of G-CSF and G-CSFR were detected by RT-PCR, Western blot and immunohistochemistry. The results of RT-PCR showed that mRNA of G-CSF and G-CSFR was expressed by all four grade gliomas. The results of western blot and immunohistochemistry showed that protein of G-CSF and G-CSFR was also expressed by all four grade gliomas. Co-expression of G-CSF and G-CSFR was detected in7/8Ⅰ/Ⅲ gliomas,8/9Ⅲ anaplastic gliomas and12/12Ⅳ glioblastomas. The differences of expression levels in four grade gliomas were not significant. Expression of G-CSF and G-CSFR was not detected in the5normal brain tissues.We also detected the expression of G-CSF and G-CSFR in primary cultured glioma cells G0518, glioma cell line T98G and primary cultured astrocytes. The results showed that G-CSF and G-CSFR were co-expressed by G0518and T98G, however, not expressed in primary cultured astrocytes. These results indicated that G-CSF signaling may play a role in the progression of glioma.2. Influences of G-CSF on proliferation, migration and invasion of glioma cellsTo further evaluate the role of G-CSF signaling in glioma, we established primary cultured glioma cells G0518, glioma cell line T98G and primary cultured astrocytes. Exogenous G-CSF was used to stimulate glioma cells. MTT was employed to test cell viability, and the results showed that G-CSF obviously increased cell viability of glioma cells in a dose-dependent manner, with100ng/ml exhibits most effect function. BrdU assay, wound-healing assay and transwell assay were employed to test cell proliferation, migration and invasion, and the results showed that exogenous G-CSF promotes proliferation, migration and invasion of glioma cells. These results suggested that activation of G-CSF signaling contributes to progression of glioma.3. Mechanisms of G-CSF function on proliferation, migration and invasion of glioma cellsTo explore the mechanisms that G-CSF function on proliferation, migration and invasion of glioma cells, G-CSFR neutralizing antibody was used. The results showed that G-CSFR antibody inhibits proliferation, migration and invasion of glioma cells, and also, induces apoptosis. These results indicated that G-CSFR mediated G-CSF function on glioma cells.It is reported that Signal Transducer and Activator of Transcription3(STAT3) pathway can mediate G-CSF signaling to nucleus, thus, we evaluated the activation of STAT3following G-CSF signaling activation or inhibition. The results showed that exogenous G-CSF activates STAT3in a time dependent manner, while G-CSFR antibody inhibits G-CSF-induced STAT3activation. These results demonstrated that G-CSF signaling regulates STAT3activation.To further reveal the role of STAT3pathway in G-CSF signaling, we checked the regulation of G-CSF signaling on expression of STAT3downstream factors. The results of Real-time PCR showed that exogenous G-CSF promotes expression of Bcl-2associated X protein (Bax), B cell lymphoma/leukemia2(Bcl-2), vascular endothelial growth factor (VEGF), matrix metalloproteinase2(MMP2) and MMP9; while G-CSFR antibody displays opposite effects, and increases the value of Bax/Bcl-2. These results indicated that STAT3pathway is involved in the G-CSF signaling regulation of glioma cells.In the present study, we have shown that G-CSF and G-CSFR are over-expressed in glioma, the differences between different grades of gliomas is not significant. G-CSF increased proliferation, migration and invasion of glioma cells, indicated that it may involved in progression of glioma. G-CSFR antibody inhibited proliferation, migration and invasion of glioma cells, suggested that G-CSFR may be a potential target in glioma therapy. Furthermore, activation of STAT3and expression of its target molecular were regulated by G-CSF, which indicated that STAT3pathway is involved in the G-CSF regulation of glioma cells.Part Ⅱ:Effects of melatonin on proliferation, migration and invasion of glioma cells1. Influences of G-CSF on proliferation, migration and invasion of glioma cellsTo investigate the effects of melatonin on proliferation of glioma cells, MTT assay was employed. The results showed that neither physiological concentration nor pharmacologic concentration of melatonin affected the mitochondrial respiration in glioma cells. Trypan blue staining revealed that at concentrations1nm and1mm, melatonin did not induce glioma cell death under the present experimental conditions. We next evaluated the effects of melatonin on migration and invasion of glioma cells. Wound healing assay and Transwell invasion assay showed that melatonin significantly reduced migration and invasion of glioma cells.2. Mechanisms of G-CSF function on proliferation, migration and invasion of glioma cellsFirst of all, we evaluated the effect of melatonin on MMP2and MMP9expression. The results showed that1mM melatonin significantly inhibited MMP2and MMP9expression. These results indicated that MMPs is involved in melatonin inhibition of migration and invasion of glioma cells.Melatonin is free radical scavenger, and we have found that there was ROS production, which can be reduced, in glioma cells. These results suggested oxidative pathway may be regulated by melatonin in glioma cells. NF-kB, which can be activated by ROS and regulates production of MMPs, is one of key molecular in oxidative pathway. Thus, we studied activation of NF-kB. The results showed that there was persistent activation of NF-kB, which can be inhibited by melatonin, in glioma cells. PDTC, a NF-kB inhibitor, displayed functions similar to melatonin. These results indicated that oxidative pathway is involved in melatonin regulation of glioma cells.In the present study, we have shown that1mM melatonin inhibited migration and invasion of glioma cells. Melatonin reduced ROS production and NF-kB activation. PDTC, a NF-kB inhibitor, displayed functions that similar to melatonin. These results suggested that ROS/NF-kB/MMPs pathway mediates melatonin regulation of migration and invasion in glioma cells.