Recent reports demonstrated that stem cell tropism for tumor mediated by different kinds of tumor development inducers that concluded growth factors, chemokines, extracellular matrix proteins, transcription factor and receptors (An, 2008; Rigg, 2001; Rosenzweing, 2006; Ziu, 2006). These factors help most of tumor metastasis included glioma cell invasion that many infiltrated cells from concentrated glioma cell mass to normal brain tissues and surviving in not enough nutrition and oxygen supply condition. Even many stem cell migration inducing factors were founded, migration mechanism information rarely known. Additionally, previous experiments about stem cell based cancer therapy limited delivery of tumor growth inhibitory factor or improve of stem cell migration ability (Brown, 2003; ehtesham, 2002;
ehtesham, 2005; Tang, 2003; Tsupykov, 2009; Zhang, 2008; Zhao, 2008). In this study, identified tumor specific stem attract molecules and researched its role in stem cell migration induction and mechanism of stem cell tropism for tumor that could use for new cancer therapeutic strategy.
My results showed that selected neural stem cell attract molecule candidates using microarray were related with tumor development. Among that genes highly expressed in most tumors and make tumor surroundings for tumor growth suitable condition that induced cell proliferation, promoted tumor cell invasion and activated angiogenesis. In tumor grow condition, much oxidative stress were made by insufficiency of blood supplies to tumor center. So, it play role as anti-oxidative action (Hoehn, 2003). Periostin, osteoblast specific
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factor-2, first found in osteoblast of bone, participated in osteoblast differentiation and adhesion (Horiuchi et al, 1999). It was known as secreted protein and frequently over-expressed in various types of cancer such colon cancer (Bao et al., 2004), breast cancer (Shao et al., 2004), pancreatic cancer (Baril et al., 2007), oral cancer (Siriwardena et al., 2006), non small cell lung cancner (Sasaki et al, 2001). Furthermore, damaged heart tissues increased periostin expression level that promoted endothelial cell differentiation and migration in damaged sites (Lindner, 2005). Similarly tumor development related gene role, periostin participated in tumor metastasis, invasion, epithelial mesenchymal transmission and survival (Kanno, 2008).
Followed microarray data, periostin was commonly over-expressed in three different tumor tissues and ranked high position that highly expressed genes list in each tumor. However, real time PCR and IHC assay results showed different date compare microarray it. Periostin not highly expressed in low grade tumor such as meningioma or adenoma but did in high grade malignancy tumor like glioblastoma and astrocytoma. Periostin has malignant tumor specific expression pattern and may important act in high grade tumors. Among the people of received tumor remove surgery, benign tumor patients survival rate was high than malignant tumor type patients. Because malignant glioma cells have very aggressive character and openly made glioblastoma multiform, recurrent rate was very high and survival rate is low (Louis, 2007; Jemal, 2003). It means that identifying of malignant tumor specific gene was important in cancer therapy and periostin could be one of them.
This study focused on stem cell migration induction by periostin that promote tumor cell infiltration to tumor surround area. It could be powerful stem cell attract molecule than other
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well known factor of it. To checked stem cell migration activation, in vitro boyden chamber assay and in vivo migration assay were performed. In vitro assay result, stem cell moved to periostin solution contained bottom chamber as concentration dependent manner and picked 10ug/ml periostin concentration. In vivo result appeared that transplanted stem cells moved to p-NIH3T3 cells injected site in ipsi-laterally or contra-laterally transplanted group but not move in NIH3T3 cells injected group. As like that tumor cell responded to periostin and invaded to normal tissues, stem cells reacted to periostin and migrated to p-NIH3T3.
Specifically, periostin induced stem cell migration capacity was noticeably increased compare VEGF. VEGF was up regulated in highly vascularized tumor that malignant brain tumors than normal brain and promoted stem cell migration in vitro and in vivo conditions (Schmidt et al, 2005; Zhao et al, 2008). In vivo results, NSCs attraction by periostin connoted very important fact. In normally, NSCs has tropism for tumor that secreted many various chemoattractive molecules. Each signal of these factors combined and give a powerful cue to NSCs for tumor tropism. However, only periostin used for NSC tropism and intensely promoted NSC migration. It means that malignant tumor specific producing factor (periostin) could be key molecule of NSC tropism for tumor.
In cell migration, many kinds of extracellular matrixes (ECM), collagen, vitronectin, tenascin, fibronectin and laminin, (Goldbrunner et al, 1996; Ziu et al, 2006) were mediated through different type of integrin complexes (Bao et al., 2004; Baril et al., 2007; Yan et al., 2006) that was associated with cell adhesion, cell survival, proliferation and migration through intracellular signal cascades (Saito et al, 2010; Yamashita et al, 2010). Also, tumor produce factor induced cell migration mediated by unique receptor and various integrin
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complexes about each factors. When murine neuronal progenitor cells migrate to tumor by SDF-1alpha, CXCR4 receptors is mainly used for cell signal transduction with integrin alpha-4 (Allport et al, 2004). SDF-1 alpha regulated cell adhesion capacity that important role in cell migration using integrin VLA-4 (a4b1) up regulation in hematopoietic progenitor cell (Hidalgo et al, 2001). Netrin-4 and laminin gamma1 subunit controls neural stem cell migration in rostal migratory stream (RMS) during neurogenesis of murine through activation of integrin a6b1 mediated cell signal pathway (Staquicini et al, 2009).
Specially, periostin bind to integrin avb3 (Bao et al, 2004; Butcher et al, 2007), avb5 in ovarian cancer cells (Gillan, 2002) and a6b4 complexes in pancreatic cancer cell (Baril et al, 2007). In cell migration, Integrin aVb3 complex was mainly used that demonstrated with disrupted stem cell migration using avb3 and avb5 antibodys in in vitro migration assay (Gillan, 2002; Grzeszkiewicz, 2001). Additionally, increased periostin overexpressing 293T cell motility was blocked by integrin avb5 antibody or EGFR kinase inhibitor (Yan et al, 2006). In our system, Microarray and RT-PCR results showed that integrin beta subunits were not expressed except b1 and b5 in neural stem cells. Integrin av could not combinded with integrin b3 subnit. It suggest that periostin induced neural stem cell migration may regulated by integrin aVb5 complex and this integrin expression pattern changed by cell type.
Periostin induced tumor metastatic growth and invasion was started from focal adhesion kinase(FAK) signal pathway that activate phosphoinositide 3 kinase (PI3K) signaling (Baril et al, 2007). Actually, integrin complex has FAK binding domain that used downstream signal transduction trigger. FAK signaling was connected with various downstream pathway
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messengers. In current study, periostin induced stem cell migration is regulated by FAK mediated intracellular signal transduction (Kendall, 2008). Indeed, many tumor cells were used FAK signaling pathway with diverse downstream signal transduct proteins that depend on cell type for stem cell motility mechanism. To found FAK mediated downstream signal pathway relative protein, ERK inhibitors (PD98059) and CDK5 inhibitors (roscovitine), known as stem cell migration regulator during neurogenesis in brain (Jessberger, 2009;
Tanaka, 2004; Xie, 2004), were treated to NSCs. both inhibitors reduced periostin induced stem cell migration level to 90% and 40% compare control group as dose dependent manner.
In periostin treated NSCs, DCX was concentrated to near nucleus compare widely spread out entire cytoplasm in periostin non treated condition or VEGF treated. Nucleus translocation was important in cell migration. Cell migration began cytoplasm branch spread and finished Nucleus translocation that controlled by DCX. Besides, PAK1 and Nudle1 help this behavior.
Both protein play important role in cytoskeleton rearrangement using actin dynamics.
Western blotting results support that. Periostin lead to activation of integrin linked FAK.
Activated FAK signaling conducted to CDK5 and its downstream signal that directly regulate cytoskeleton components. In cell migration, integrin liked FAK signaling may control motility. Specially, Peirostin use CDK5 downstream signal pathway (DCX, PAK1 and Nudel1).
Until the present, many researcher develop diverse stem cell based cancer therapeutic strategies that include antibody (HER2), antitumor factor (IL-12, IL-23, interferon-b, TRAIL) (Dickson et al, 2007; Ehtesham et al, 2002; Hingtgen et al, 2008; Shah et al, 2005;Yang et al, 2004; Yuan et al, 2006), prodrug activating enzyme coding genes (CD, CE,
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TK) (Danks et al, 2007; Joo et al, 2009; Li et al, 2007) introduced stem cells (Frank et al, 2009; Mercapide, 2010). These factors have good effect to inhibit tumor growth and expand experimental animal life time. However, above trials was based on stem cell tropism for tumor. Actually each factor has limitation of apply to in vivo cancer therapy. Many antitumor factors and prodrug activation enzymes have no targeting ability that effectible factors reach from original expressed location to tumor. Even antibody, the unique antigen binding properties, has limitation of distribution to deeply existing tumor cells and prevented infiltration to it by blood brain barrier (BBB). So far, stem cells use as vehicle for antitumor agents delivery with properties of tumor targeting and infiltration to deep inside tumor mass.
Additionally, stem cells are able to cross the BBB. It makes that increasing of antitumor efficacy to tumor.
This study was designed to improve stem cell based cancer therapy using tumor derived stem cell attract molecule and tumor growth inhibition gene or suicide gene encoding stem cell. In bystander effect in vivo assay results using CD-HB1.F3 with 5-FC, rat brain tumor size were significantly decreased by 60%. It correspond well with those of the earlier study which reported that tumor invasion and growth suppression gene having stem cells can reduced increasing tumor size and expanded animal survival. However, in case of the tumor growth speed is too high, even therapeutic gene having stem cell on reach tumor growing position could not play tumor repression function (Danks et al, 2007; Joo et al, 2009). If more specific gene carried stem cells are more rapidly move to tumor developing site, cancer therapeutic efficacy will be increased. In present study, increased amount of periostin promoted stem cell migration rate and tumor growth inhibition rate followed periostin
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concentration. Tumor size decrease was caused by prohibited infiltration from injection site to near normal tissues. This result is in contrast to previously report study that periostin induced tumor metastasis. However, in this system, periostin was not affected to giloma growth and migration. It supported by in vitro cell co-culture examine using boyden chamber.
C6 rat glioma growth rate not differ between C6 cell co-culture condition and p-NIH3T3 did (Supplement. 1). It means that periostin could regulate stem cell tropism for tumor cell very efficiently. I demonstrate that cancer therapeutic efficacy of F3-CD cells could increased by periostin promoted cell migration ability improvement.
In summary, malignant tumor originated periostin serve as a stem cell migration regulator, with FAK and CDK5 signal transduction through integrin aVb5 complex, for tumor repression gene delivery system on cancer therapy. Present study propose that periostin may play a crucial role in establish strategy for stem cell based cancer therapy.
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