FAK and CDK5 signal pathway was included in the neural stem cell migration 44

integrated integrin complex. FAK signaling pathway well known as cell motility related signal pathway. FAK signal pathway connected with many kinases in its downstream and convey signal. In cell migration, PI3K / AKT signal pathway was mainly used that located in FAK downstream. So, i checked activation of many kinases included in FAK downstream signal pathway. To research kinase activation, Cell lysates were collected from periostin treated NSCs as time increasing manner in ranging from periostin non treated condition to 12hr treated and detected with phosphorylate form kinase antibody.

At first, i observed FAK activation level that first trigger of signal transduction from ligand (periostin) to inside of cell. Western blot results showed that FAK intensely activated at periostin treat 30 minutes after time point. AKT and mTOR were activated followed FAK at periostin treat 1 hour time point. Subsequently, i researched CDK5 activation that controlled by mTOR and well known regulator of Nudel1 and PAK1. Both proteins control cytoskeleton component arrangement. Western blotting result showed that CDK5 activation appeared on periostin treated one hour later and maintained until periostin treat 12 hours time point. Coincidently, PAK1 activation started at 1hr time point and continued 6hr.

Nudel1 also activated at PAK1 activation decreased time point and continued 12 hours as more activated pattern. After all, periostin induced stem cell migration signal was madden with FAK signal pathway that conveyed to AKT signaling and CDK5 downstream signal pathway.

45

Con 30min 1hr 3hr 6hr 12hr

ERK adjusted as equal amount and performed western blotting. Protein activation related with periostin induced NSC motility was detected using each antibody as phosphorylated form.

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8. Neucleokinesis in neural stem cell migration by periostin

Included with Nudle1 and PAK1, DCX control cell migration using actin and tubulin rearrangement. In central nervous system development, NSC moved from subventricular zone to cortex through radial glial cell using ERK and CDK5 signal pathways. In neuronal migration, DCX participated in nucleus translocation, neucleokinesis, (Niethammer et al, 2000). Like this, whether periostin induced stem cell migration related with these pathways, i investigated ERK and CDK5, specifically DCX, involvement in periostin induced cell migration with in vitro migration assay and immunocytochemistry.

Like above results, periostin induce NSC migration. When ERK and CDK5 inhibitors were treated with periostin to NSC, periostin induced NSC migration was weakly interrupted by ERK inhibitor PD98059 (40% cells of migrated stem cell). However, Roscovitine, CDK5 inhibitor, significantly repressed periostin induced hNSC migration (94% cells of migrated stem cell) and responded dose dependently (Fig. 12A). These results elucidate that periostin induced NSC migration was controlled by CDK5 and ERK. And CDK5 intensely regulate cell motility than ERK. Because of DCX, controlled by CDK5, concentrated to near nucleus when they participate in nucleokinesis, I observed DCX location changing in periostin treated cell using immunocytochemistry method. In situation of induced NSC migration by VEGF or periostin, ERK location pattern changing was not observed in both conditions. Also, DCX location was not changed in VEGF treated NSC but noticeably changed in periostin treated condition (Fig. 12B). Widely spread DCX in cytoplasm move to near the centrosome in activated cell migration condition. This phenomenon was interrupted with roscovitine.

With these results, i assumed that periostin promoted NSC migration was used neuronal

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migration signaling pathway. Specifically, CDK5 involved nucleokinesis was mainly participated in periostin induced NSC migration with weak ERK signaling pathway.

48

B

DCX

ERK

Fig. 12. Periostin promoted NSC migration controlled by CDK5 signal pathway. (A) Periostin induced NSC migration inhibit assay were examined with ERK inhibitor (PD98059) and CDK5 inhibitor (Roscovitine) that respectively treated to NSCs as dose increased manner. periostin promoted NSCs migration was significantly interrupted with increased roscovitine concentration. (B) DCX and ERK location change in periostin induced NSC migration. In vitro migration assay and immunocytochemistry was performed to detect DCX and ERK location in periostin (10mg/ml) or VEGF (10ng/ml) treated NSCs. Periostin and VEGF induce cell migration. In periostin treated condition, DCX concentrated to near the nucleus

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(small box) and interrupted by CDK5 inhibitor (Roscovitine). However, VEGF is not change DCX and ERK location. Scale bar : 50mm

50 9. Cytotoxic effect of F3-CD cell to p-NIH3T3 cell

To investigate whether suicide gene encoding NSC deliver bystander effect to tumor, I constructed cytosine deaminase (CD) expressing HB1.F3 (F3-CD) cell line and checked the expression of CD gene using RT-PCR (Fig. 13A). Enzyme activity assay and HPLC analysis was conducted to confirm that CD protein activity of conversion prodrug 5-fluorocytosine into the cytotoxic 5-fluorouracil. The supernatants of the F3-CD cell cultures with 5FC showed two peaks, identified as 5FC and 5FU by comparison with the standards. F3-CD cells converted FC to 5FU as 93mM in cultured medium (Fig. 13 C and D). Additionally, 5-FU of 2.9mg was produced by Cytosine deaminse that examined with 50 mg F3-CD cell lysates in the presence of 1mM 5-FC for 8hours (Fig. 13B). It means that CD gene encoding F3-CD cells were produced normal enzyme activity having cytosine deaminase and converted 5-FU secreted to cultured medium continuously. Secreted 5-FU has important role in bystander effect to neighborhood cells.

I evaluated the F3-CD cell cytotoxic effect in C6 cells or coculture system with F3-CD cell in 5-FC presence condition. 5-FC treated 3day after, survived C6 cells was counted and quantified compare only C6 cultured condition. More F3-CD cells cocultured with C6 cell, cytotoxic effect was increased and 80% of C6 cells are death at 5% F3-CD cells to C6 cell was mixed condition (Fig. 14). Additionally, i examined in vivo bystander effect assay. F3-CD cells transplanted to p-NIH3T3 cell bearing animal that receive 5-FC of various concentrations as dose increasing manner daily for 2 weeks. Periostin over-expressing NIH3T3 cell survival rate was stereologically calculated. In F3-CD cell transplanted with higher 5-FC concentration animal, survived p-NIH3T3 cells noticeably decreased (44% of

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pNIH3T3) compare F3 cell transplanted model (Fig. 15). These results suggest that constructed F3-CD cell lines have bystander effect capacity and applicable to in vitro and in vivo cytotoxic effect experiments.

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Fig. 13 . Expression of CD gene and enzymatic activity of CD in F3 and F3-CD cells.

(A) Expression of cytosine deaminase gene in CD gene transduced F3 cells and normal F3 cells. (B) In vitro total CD enzyme activity with 50 mg proteins of cell lysates in the presence of 1 mM 5-FC for 8 hours. (C) HPLC tracings of the supernatants from F3-CD cell culture medium for 2 days are shown. UV absorbance (270 nm) is shown on the ordinate; retention times of various standards are indicated on the top. (D) Quantification of HPLC expressed as percentage of maximum peak for each metabolite.

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Fig. 14. Suicide gene (cytosine deaminase) encoding NSCs induced neighborhood cell death. (A)C6 cells were cocultured with F3-CD cells at various ratio (0-200% cell versus number of cultured C6 cell). One day after, every groups, except C6 alone cultured group, treated with 5-FC for 3 days. (B) Survive cells were counted and evaluated compare only C6 cells cultured condition. Survival rate of C6 cell was more decreased with increased number of F3-CD cell. All experiment were independently conducted and appeared as means ± SD. Scale bar : 100mm.

54 5-FC

250 mg/kg 500 mg/kg 1000 mg/kg

F3 death of p-NIH3T3 was increased by transplanted F3-CD cells with systemic treated 5FC. Suicide gene transduced NSCs injected to p-NIH3T3 implanted animals. NSC injected after 5 days, animals received 5-FC of variety dose daily for 2 week through

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interperitoneal injection. (A): Fluorescence image through p-NIH3T3 cells taken at increasing 5-FC dose after F3-CD cell implanted. (B): Graph represents quantification of p-NIH3T3 cell number in each 5-FC dose treated rat model using stereology investigator program. Bystander effect of F3-CD in vivo was significantly showed at 500mg/kg concentration of 5-FC treated group. Green: Dycycle green labeled p-NIH3T3 cells. Scale bar : 100mm. T-test : ** = p<0.025, *** = p<0.01.

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10. The therapeutic effect of F3-CD cells in rat glioma model.

In above results showed that the migration potential of NSCs was induced by periostin.

Much more NSCs having therapeutic agents moving to cancer cell could be giving very efficient anticancer effect. I demonstrate that F3-CD cells affected to neighborhood cell as induced cell death. In vivo bystander effect experiment performed to assess the clinical efficacy of F3-CD in C6 rat glioma animals that receive rat glioma cells with p-NIH3T3 cells.

Wide range of p-NIH3T3 cell number regulated F3-CD cells motility capacity. For same period, Number of chased F3-CD cells to C6 glioma cells was largely expanded as much p-NIH3T3 cells injected with glioma cells (Fig. 16A). Similarly with these result, tumor growth of F3-CD transplanted animal was remarkably inhibited (40% of tumor size) compare only C6 implanted animals. Beside, tumor size was decreased p-NIH3T3 cell number dependently that increased F3-CD cell number of reached to tumor mass. In same number of p-NIH3T3 cell implanted animals, more time permitted F3-CD cells present noticeable decrease of tumor size (60%) compare little time permitted it (Fig. 16B). The decreased glioma size looks like that caused by inhibition of C6 glioma cell invasion. In C6 alone transplanted group, C6 cells scattered near the original transplanted site. However, widely infiltrated C6 cells did not observed in more p-NIH3T3 cell injected groups (Fig. 16).

In heamtoxylin and eosin staining results supported that C6 rat glioma cell infiltration was inhibited (Fig. 17). Inhibition of glioma cell infiltration was reinforced by increasing p-NIH3T3 and expanded period of F3-CD cell migration.

It results suggest that suicide gene encoding F3 cells very efficient method to reduce tumor cell growth. The more F3-CD cell migrated to cancer cell, the more cancer therapeutic

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effect was observed. In these condition, continuously expressed and secreted periostin could improve therapeutic effect through induce F3-CD cell mobile ability.

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Fig. 16. Therapeutic effect by systemic F3-CD treat with 5-FC on C6 rat glioma cell.

DiI labeled F3-CD cells were contra laterally transplanted into C6 rat glioma cells

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only (A) or injected with various number of p-NIH3T3 cells bearing rat brain cortex followed 5-FC treated for 2 weeks (B, C). Rat brain slices observed at 5-FC treat finished time point. Rat glioma cell number and tissues size in each group were calculated compared only C6 cell injected rat model using stereology investigator program (D). F3-CD combined with periostin has stronger bystander effect to tumor growth with periostin releasing migrating time increase. Red: DiI labeled NSCs, Green: Dyecylce green labeled C6 rat glioma cell. Scale bar : 100mm. T-test : ** = p<0.025, *** = p<0.01

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F3CD (10day) C6 +

P-NIH3T3 (50%)

C6 +

P-NIH3T3(100%) C6 + P-NIH3T3(200%)

B

F3CD (10day) C6 +

P-NIH3T3 (50%)

C6 +

P-NIH3T3(100%) C6 + P-NIH3T3(200%)

A

Fig.17. C6 rat glioma cell invasion was inhibited by systemic treated 5-FC with F3-CD cells. Rat brain slices obtained from p-NIH3T3 cell mixed rat glioma model that

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examined using different migration period, 7days (A) or 10 days (B), premised F3-CD cells with 5-FC treatment for 2 weeks. Each slice was stained using Hemotoxyline and counter stained with eosin solution. Increasing periostin releases clear the scattering glioma cells around the original transplanted site. Scale bar : 100mm.

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Ⅳ. Discussion

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

Additionally, stem cells are able to cross the BBB. It makes that increasing of antitumor