Differential expression of 14 Candidate genes in several human brain tumor tissues

Ⅲ. RESULTS

2. Differential expression of 14 Candidate genes in several human brain tumor tissues

and related to tumor metastasis, growth and cell migration. To confirm the candidate genes expression pattern, I examined real time PCR in 11 different kinds of tumor that classified benign and malignant tumors (Table. 3). Those candidates showed very various expression patterns. Most genes –CXCL10, FN1, GLIPR1, GPX3, PROS1, SFRP4, TIMP1, TIMP2, TNFRSF1A, TNFSF13B - were commonly over-expressed in each tumor. Those genes may play same role in most tumors that indistinct tumor group. TNC appeared any tumor specific expression pattern. However, POSTN was showed very different expression pattern. It was highly expressed in malignant tumors but not in benign tumors. It could be participated in malignant tumor development mainly (Fig. 3). Additionally, POSTN gene expression manner in real time PCR data matched with microarray data that presented commonly over-expressed in 3 different brain tumors. Those tumors have malignancy character. This result support that periostin could important factor in tumor development especially malignant tumor. Its discrimination with other genes was important to choose candidate for malignant targeting cancer therapy.

Table. 3. Information of tumors used in real-time PCR assays

Sample Gender Age Histology

Tumor1 F 59 adenoma

Tumor2 F 56 adenoma

Tumor3 F 36 Choroid plexus papilloma

Tumor4 M 36 meningotheliomatous meningioma

Tumor5 F 48 meningioma

Tumor6 M 45 Fibroblastic meningioma

Tumor7 F 46 Fibroblastic meningioma

Tumor8 F 56 meningioma

Tumor9 M 63 schwannoma

Tumor10 M 45 Anaplastic oligodendroglioma

Tumor11 M 33 Glioblastoma

25 different human brain tumor tissues. Expression of 14 genes was analyzed by real-time RT-PCR, and fold changes in expression are expressed as the ratio of GAPDH-normalized brain tumor tissue/normal brain tissue values. Asterisk indicates the periostin, the only gene specifically expressed in malignant tumor samples.

3. Periostin more intensely expressed human brain tumor tissues than normal brain Periostin usually expressed in osteoblast cells and found to be overexpressed in various types of human cancer, such as non-small-cell lung carcinoma, breastcancer, colon cancer, head and neck cancer, ovarian cancer, and pancreatic ductal adenocarcinoma. Whether brain tissues expressed periostin in protein level, i carried out immunohistochemistry with periostin antibody in normal or tumor tissues. In normal brain tissues, periostin was very weakly expressed compare tumor tissues intensely expressed (Fig. 4 C-E). Periostin was expressed broadly in entire tumor tissues but not all tumor cells expressed it. Furthermore, periostin expression manner was changed followed tumor. Low grade tumor (meningioma) rarely expressed periostin but high grade malignancy tumors (Astrocytoma and Glioblastoma) significantly expressed it (Fig. 4F). It expression pattern in tumor tissues supported by real time PCR result that periostin highly expressed mainly malignant tumor. It means that periostin could be important candidate for malignant tumor specific cancer therapy.

Normal

Tumor

B

C C’

F F’

D D’

E E’

A A’

B’

Fig. 4. GBM tumors have high periostin expression. Periostin was hardly detected in normal brain. Human brain tumor (C-F) and normal brain tissues (A and B) were fixed with 4% paraformaldehyde followed sectioned at 30mm on a cryostat. In each slices, periostin was detected using DAB staining method with periostin antibody (1:500). Slices of normal brain (A-B) or low grade tumor (F) presented rarely expressed periostin. However, Periostin intensely expressed only malignant tumors (C-E). Scale bar, Left panels (A-F) : 500mm, Right panels (A’-F’) : 100mm.

4. Periostin strongly attracted neural stem cell than VEGF

Whether periostin attract human neural stem cell, i checked migrated NSCs number with boyden chamber assay. Cultured NSCs in transwell upper chambers transferred to periostin treated lower chamber. 12 hours after, we counted migrated NSCs that passed boyden chamber membrane from upper position of membrane to periostin contained low chamber.

NSCs motility was promoted by periostin dose dependently about 3 times than periostin no treated condition. Also, periostin induced NSC attraction effect was picked at 10ug/ml periostin concentration but higher concentration decreased its effect. (Fig. 5A spot line)

To confirm stem cell attraction capacity by periostin, i compared periostin to VEGF which used as positive control. VEGF was highly expressed factor in cancer and have angiogenesis ability. Besides, it was well known as cell attract molecule. Various stem cell lines controlled by VEGF in migration procedure. VEGF induced NSC migration about 50%

compare VEGF no treated condition but periostin induced about 100%. Periostin has 2 times NSC attraction ability than VEGF (Fig. 5A). Compared two factor results indicated that NSC migration ability was strongly increased in periostin treated condition compare VEGF treated it. Additionally, i experiment cell type motility discrimination between HB1.F3 and HB1.F5 that has strong tropism for tumor. In VEGF treated same condition, both cell lines showed same migration rate in dose dependent manner. However, they showed difference of stem cell attraction ability in comparison with VEGF and periostin. When both factors were treated for same time, HB1.F3 cells migration ability was increased 92% by periostin and VEGF increased 26%. Its discrimination rate is about 3 times. Even HB1.F5 has no difference by both factors (Fig 5 A and B). It means that periostin could be powerful neural

stem cell attraction molecule than others and very unique to HB1.F3.

And i checked that NSC motility about periostin treated time. NSCs were cultured in transwell upper chambers for 12hr followed transferred to periostin contained lower chamber.

In each time point (4-24hours), i counted migrated NSCs from upper chamber to periostin contained low chamber. The number of migrated NSC was increased as time passes in periostin treated condition (Fig. 6 A and B). Periostin treated 12 hour after, the number of migrated NSC was noticeably increased and continued 24 hour time point. To confirm that cell migration pattern by different cell type, i used two types of NSC (HB1.F3 and HB1.F5) (Fig 6 C and D). Both cell lines showed not difference in cell migration by periostin. In summary, periostin promote NSC migration as time and concentration dependent manner and show very powerful attraction of NSC that more unique in HB1. F3 cell line.

50 100 150 200 250 50 100 150 200 250

0 5 10 20

Number of m

i

grated cellNumber of m

i

grated cell VEGF (ng/ml)

POSTN (mg/ml) HB1.F3

VEGF (ng/ml)

POSTN (mg/ml) HB1.F5

concentration

A

B

Fig. 5. Periostin significantly promote NSC migration ability compare with VEGF.

NSC chemotactic motility measured using in vitro migration assay using boyden cahmber. NSCs (5 x 10⁴ cells) inoculated to upper chamber. 1 day later, upper chamber transferred to VEGF or POSTN contained low chamber. VEGF or POSTN of different concentration were applied to HB1.F3 cells (A) or HB1.F5

cells (B) as concentration rising pattern. 12 hours after, migrated NSCs were stained with hematoxylin and counted. Black continues line indicated VEGF induced NSC migration level and spot line indicated POSTN that. POSTN promoted migration of F3 cells 3 times higher potency. However, F5 cells showed similar migration induction by both factors. The results represented as means ±SD

33 cells was inoculated in transwell upper chamber and cultured for 1day. After then, upper chamber transferred to lower chamber filled with VEGF (10ng/ml). Migrated upper side to lower side of upper transwell HB1.F3 cells was stained with H&E and counted at each time point (B). HB1.F5 cells tropism was performed as same method. (C), H&Estain result. (D), Migrated NSCs was calculated. Both neural stem cell lines showed same migration pattern that NSC migration capacity was increased time dependently. Scale bar : 200mm

5. Neural stem cell has motility to Periostin expressing NIH3T3 in vivo condition To check NSC migration activation by periostin in vivo condition, i constructed periostin over expressing NIH3T3 cell line (p-NIH3T3) using retroviral vector system (Fig. 7 A and B). Periostin gene and amphotropic envelop gene encoding were confirmed with enzymatic gene restriction assay. Periostin gene transduce cells were selected by blastidine S contained medium culture for 2 week. We confirmed periostin expression and release to culture medium with ELISA method and western blotting (Fig. 7 C and D). The p-NIH3T3 cell line (1-8) that most intensely expressed a periostin than others was chose for further studies.

Whether NSC has tropism for periostin over expressing NIH3T3 cells, i transplanted p-NIH3T3 cells to rat right hemisphere and followed NSC transplantation to same rat hemisphere cortex on 5 day. 2~4 weeks after, we removed rat brain and observed NSC tropism for NIH3T3 cells in vivo condition. NSC migrated from transplanted site to p-NIH3T3 injected position passed corpus callosum and infiltrated to p-p-NIH3T3 cell. It looks like that p-NIH3T3 cells were raped by NSCs (Fig. 8 A and B). Indeed, many NSC cells reached and posited near the p-NIH3T3 cells in injected original site (Fig. 8 C and D). This situation was not raised in NIH3T3 cell transplanted environment. NSC migrated with any direction or located in original transplanted site.

NSC mobile routes were similar to each cell inject needle track. In procedure of each cell were injection, needle could madden tunnel in brain tissues that may be used by NSC for their efficiency. Additionally, i observed that p-NIH3T3 expression was discontinued for long time using immunohistochemistry assay with periostin antibody in the experiment of NSC migration condition for 4 weeks. To avoid possibility of mechanical support by needle

and periostin discontinuously expression, i performed modified in vivo model experiment that P-NIH3T3 cells were injected to right hemisphere and NSCs were transplanted to left hemisphere cortex. 1~2 weeks later, rat brains were removed and observed. Like to NSC migration pattern in ipsi-lateral transplantation condition, NSC chased to P-NIH3T3 cell followed corpus callosum route that connect construct of right and left hemisphere and attained original P-NIH3T3 injection site and near site of it (Fig 9.). Also, periostin continuously expressed (Fig. 9 D and I).

These results suggest that periostin induced NSC tropism powerfully and give some cue about their migration direction. Also, it is possible in both in vitro and in vivo situation.

36 293T cell

pCXbsr (2455 bp) pCL-Ampho (4070bp)

1Kb DNA EcoR1 Sal1 EcoR1

Sal1 100 bp 1Kb DNA Cla1 Sal1 Cla1 Sal1 100 bp

3 Kb 10 Kb

2 Kb 3 Kb 10 Kb

2 Kb

A B

NIH3T3 cell

1-20 1-16 2-2

1-12 1-8 1-24

WB : POSTN 50 100 150 200 250

POSTN level in culture medium(%)

Fig. 7. Construction of periostin over expressing NIH3T3 cell line. 293T cell transfected with periostin gene encoding pCXbsr vector, containing the blastcidine S resistant gene, and pCL-Ampho vector which expresses an amphotropic envelope.

Transfection two days after, the culture medium collected and incubated with NIH3T3 cells. After then, cells were selected with blasticidin S (5 mg/ml). (A, B), Enzymatic gene restriction was performed to confirm that existence of periostin gene and amphotropic envelope gene in each plasmid. Among of constructed periostin overexpressing cell lines, 1-8 cell line selected using ELISA assay that evaluate secreted periostin amount to culture medium (C) and confirmed with western blotting (D).

POSTN POSTN--NIH3T3NIH3T3

2 week 4 week

B C

D E

Fig. 8. Ipsi-laterally transplanted NSCs have tropism for periostin over-expressing NIH3T3 cells. DiI labeled NSC transplanted to p-NIH3T3 or NIH3T3 implanted same hemisphere cortex. DiI labled NSC located in original injected site and showed not tropism for NIH3T3 that at time point of transplanted 2 weeks later (A).

However, NSC migrated through corpus callosum and reached Vybrant Dyecycle Green labeled p-NIH3T3 (B) that continuously expressed periostin (small box in Fig. B). NSC transplanted 2 weeks later, NSCs distributed entire p-NIH3T3 cells which spread from implanted position to near striatum tissues and intermixed with p-NIH3T3 cell (D). In more times was given to NSC condition (4 weeks), NSCs

have motility to p-NIH3T3 (C) and constantly maintained (E). In addition, periostin expressed continuously (small box in Fig. C). Coronal section diagram of rat brain indicate both cell injected site. Scale bar : (A-C), 200mm; (D,E), 50mm

F3 POSTN- NIH3T3

POSTN- NIH3T3 F3

2 week 1 week

I J

H E C

Fig. 9. Contra-laterally transplanted NSCs reached to p-NIH3T3 cell. NSC was labeled with DiI and injected contra-laterally to p-NIH3T3 cell implanted rat hemisphere cortex. Even both cells have long distance between that, DiI labeled NSC moved follow corpus callosum (C) and surrounded p-NIH3T3 cells (A, B). For 2 weeks after NSC cell injection, NSCs traveled to p-NIH3T3 cells which located far from NSC injected site and infiltrated themes (F, G) through corpus callosum route (H).

Periostin expression in vivo was confirmed using immunohistochemistry with DAB (D and I) or DAPI (E and J). Periostin continuously expressed in p-NIH3T3 implanted 1 or 2 weeks passed time point. Red: DiI labeled NSCs, Green: Dyecycle labeled p-NIH3T3 cells, Rat coronal brain section diagram showed NSCs migration route and injected site or p-NIH3T3 cell implanted site. Scale bar : (A,F), 1000mm;

(B-C, G-H), 50mm; (E, J), 200mm.

6. Periostin Receptor expression in neural stem cell lines

Periostin bind to diverse integrin complex that composited with integrin alpha subunits and beta subunits. Integrin alphaV and beta3, alphaV and beta5 and alpha6 and beta4 complexes were known as periostin receptor that related with cell migration. In cell migration, Integrin avb3 and avb5 were mainly used and a6b4 used in some cell type. We investigated that existence of integrin subunits in HB1.F3 NSC using reverse transcription-PCR and Microarray. In Microarray results, i observed that integrin alpha V subunit and beta1 and 5 subunit were highly expressed but not beta 3 and 4 (Fig. 10A). RT-PCR results showed same result in HB1.F3 NSCs (Fig. 10B). Through above results, i assume that integrin aVb5 complexes could use in NSC migration instead of aVb3 complex or a6b4.

43 NSCs using Trisol method and analyzes with Microarray. Reverse transcription PCR experiments were conducted in HB1.F3 using individual integrin subunit coding probe.

7. FAK and CDK5 signal pathway was included in the neural stem cell migration We observed that periostin promoted stem cell migration arise from FAK activation that 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.

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.

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

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

B

^Con ^VEGF ^POSTN POSTN + Inhibitor

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

(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

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.

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.

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

문서에서 Glioma produced periostin attracts human neural stem cells (페이지 35-0)