Malignant gliomas (glioblastoma multiforme and anaplastic astrocytoma) occur more frequently and lethally than other types of primary CNS tumors, having a combined incidence of 5–8/ 100,000population. (Henry et al, 2000; Louis et al, 2007) Among them, glioblastoma multiformes (GBMs) are the most frequent and malignant form of brain tumors.
(Lim et al, 2011; Munshi et al, 2009) The tumor microenvironment exhibits expression of pro-inflammatory molecules that promote migration and invasion of tumor cells. (Tafani et al, 2011; Goldbrunner et al, 1998) So, glioma can infiltrate aggressively and invasively into surrounding regions. Despite combined treatment of chemotherapy and radiotherapy after surgical excision, the median survival time after diagnosis is still in the range of just 14 months. (Smith et al, 2000; Ohgaki et al., 2004; Yamanaka et al, 2009) Moreover, although advanced in medical technology and discovery of new anticancer drugs, malignant glioma is still a serious health problem and conventional therapeutic effects on it still show poor prognosis and incurable in large proportion of patients. So, new and efficient therapy is required for malignant gliomas. In this regard, neural stem cell-based gene therapy can be regarded as a promising alternative. (SHI et al, 2009; Donghong et al, 2008; Seung U Kim, 2011)
Recent research indicates that the discovery of the inherent tumor-tropic properties of neural stem cells (NSC) can specifically target invasive solid tumors, including glioma, and thus provide a novel platform for targeted delivery of therapeutic agents to tumors and
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overcoming the primary challenge in developing chemotherapy regimens, resulting in significant antitumor effect. (Schmidt et al, 2005; Donghong et al, 2008)
To apply this strategy to the treatment of brain tumor, the tumor-tropic properties of neural stem cells (NSCs) led to the development of a novel strategy for delivering therapeutic genes to tumors in the brain. Human neural stem cells (hNSCs) possess an inherent tumor tropism that supports their use as a reliable delivery vehicle to target therapeutic gene products to primary and secondary invasive glioma cells throughout the brain, as well as to other types of solid tumors, including melanoma brain metastases, medulloblastoma, and neuroblastoma .(Kendall et al, 2008; Heese et al, 2005; Seung U Kim, 2011) Brain tumors including malignant gliomas are known to release numerous cytokines, chemokines and growth factors that are capable of stimulating the directed migration of NSCs into the tumor environment. Immortalized hNSC lines, such as HB1.F3, are particularly well suited for the expansion of clones that stably express therapeutic genes designed to treat invasive gliomas.
(Kendall et al, 2008) The novel and significant feature of neural stem cell-based gene therapy for brain tumor is that it may be viable to exploit the tumor-targeting therapy for invasive and metastatic tumors in the brain, for which no curative treatments are currently available.
A recent studies showed that NSCs have the ability to target cytosine deaminase (CD) to tumor cells, where CD can convert the systemically administrated prodrug 5-fluorocytosine (5-FC) into the toxic anticancer agent 5-fluorouracil (5-FU), which kills tumor cells in vivo by triggering apoptosis. (SHI et al, 2009; Aboody et al, 2006; Egeblad et al, 2002) Like a suicide gene therapy has been found to be effective on malignant tumors, although its
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application is very limited. (Springer et al, 2000) In here, availability of tumor targeting can elevate suicide gene therapy by CD and 5-FC.
Tumors secrete multiple growth factors that drive activation, migration and proliferation of vascular endothelial cells, including TGF-α, bFGF and VEGF. (Folkman et al, 2003) CM-1, also named osteoblast-specific factor 2, is a kind of bone adhesion molecule that regulates osteoblast adhesion and differentiation and is classified among the extracellular matrix (ECM) proteins. (Wang et al, 2012) The sequence of CM-1 contains a typical signal sequence, a cysteine-rich domain, a fourfold fasciclin 1- like (FAS-1) domain and a C-terminal domain. (Horiuchi et al, 1999; Takeshita et al, 1993) Recently, the overexpression of CM-1 has been found in various human cancers including non-small cell lung cancer, ovarian cancer, breast cancer, colon cancer, pancreatic cancer, liver cancer, oral cancer, head and neck cancer and neuroblastoma. (Shao et al, 2004; Zhu et al, 2010; Bao et al, 2004;
Kudo et al, 2006; Baril et al, 2007; Riener et al, 2010) CM-1 is reported to be involved in tumor EMT, extracellular matrix degradation, tumor invasion, and distant metastasis, but the mechanism by which it operates is still unclear. (Wang et al, 2011; Morra et al, 2011)
CM-2 is multifunctional proteins that can inhibit the catalytic activity of MMPs, thus maintaining ECM homeostasis. Besides acting as proteinase inhibitors, TIMPs also modulate cell growth, apoptosis, and angiogenesis. (Lambert et al, 2004) Recent transgenic studies also showed that endogenous overexpression or suppression of CM-2 inhibited or enhanced the tumor growth in several cell types, respectively. (Martin et al, 1996; Kruger et al, 1998 and Martin et al, 1999) The CM-2 field was complicated in recent years by the finding that CM-2 is often overexpressed in many malignancies, including lung, ovarian and breast
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cancer. (Egeblad et al, 2002) In addition, CM-2 has been shown to exert an antiangiogenic activity both in vitro and in vivo. (Johnson et al, 1994; Roberts et al, 1993)
Through microarray analysis, previous results showed that CM-1 and CM-2 were commonly over-expressed in tumor tissues of glioblastoma patients. First, CM-1 was proved to induce migration of NSCs in vitro/ in vivo and activated migration signaling pathway such as FAK and CDK5. In vivo, CM-1 by CM-1-producing NIH3T3 cells (p-NIH3T3) significantly induced migration of NSCs and NSC encoding cytosine deaminase (CD), suicide gene delivered bystander effect targeting to tumor more efficiently. Furthermore in vivo result showed obvious edges of tumor mass. So, we hypothesized that CM-1 was effective to assemble cancer stem cells and neural stem cells by integrin αvβ5 as well as prolonged survival time using systemic strategy.
The other chemoattractants CM-2, as prior studies, specifically led to migration of NSCs by interacting with CD63 as well as by implicating CDK5 and ERK signal pathway. Here, we determined more detail signaling mechanism whether integrin β1 and phosphoinositol-3-kinase (PI3K) signal pathways control induction of NSCs migration via CM-2. Moreover, phosphorylation of focal adhesion kinase (FAK) is regulated by CM-2 induced migration but not in CD63 knockouted NSCs.
In this study, we determined that CM-1 induced tumor-tropism of neural stem cells for brain tumor therapy along with NSCs encoding cytosine deaminase and prodrug 5-FC that resulted in effective increased survival in brain tumor models. Furthermore, we investigated functional mechanism involved in CM-2 induced in vitro mobility of neural stem cells. In the present study, we provide the evidence that CM-1 and CM-2 could serve as key molecules in
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NSCs tropism and combination with genetically modified NSCs as well as CM-1 and CM-2 strongly block the infiltration of tumor cells.
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