DISCUSSION

The present study identified CM-1 and CM-2 as novel chemoattractants molecules for mobility inducers in activating tumor tropism of NSCs. According to prior results, CM-1 and CM-2 were over-expressed in human glioblatomas tissue and possessed ability of induction of NSCs migration. Furthermore, when human NSC line HB1.F3 carrying cytosine deaminase (CD) enzyme gene (F3.CD) was transplanted intracranially at distant sites from the tumor, the NSCs migrate through normal tissue and selectively “ home in” to the glioblastoma tumor mass and upon administration of prodrug 5-FC, significant reduction in tumor volume was demonstrated in rat brain tumor model. (Kim et al, 2011; Black, 1991;

Heese et al, 2005)

In previous results, tumor mass was assembled to tumor area and non-invasive signature relatively. Cancer stem cells, one of the cells that comprise the tumor, have aggressive and invasive features are related to metastasis. So, we hypothesized that cancer stem cells had integrin αvβ5 as a receptor of CM-1 and induced cell migration via CM-1. As shown in our result, human glioblastoma primary cultured cells and C6 rat glioma cells had integrin αvβ5 and led to migration toward CM-1.

As well as in this study, we identified that use of CM-1 prolonged survival time with advanced strategy using CD and 5-FC suicide gene therapy for brain tumor model. Among experimental group D of CM-1-over expressing NIH 3T3 cells 0.5x and experimental group E of CM-1-over expressing NIH 3T3 cells 2x (compare with C6 number), tumor volume rate

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of group D was similar with group E until 30 days. But, after then, rate of tumor volume was rapidly increased and survival time was not different with control group. In contrast, the group E showed distinctly slowed the tumor growth rate as well as increased survival rate compared with other groups. It means that CM-1 of the more constant concentration was significantly effective to brain tumor treatment.

Other previous results showed that CM-2 induced migration specifically in NSCs and interacted with CD63 and integrin β1 for induction of migration. When we blocked integrin β1 using functional blocking antibody, induction of migration was significantly decreased despite the presence of CM-2. Some researchers demonstrated phosphoinositol 3-kinase (PI3K), which has been shown to be an important regulator of directed NSCs migration.

(Wozniak et al, 2004; Zhao et al, 2009) These observations revealed when CM-2 induced NSCs migration, PI3K signaling pathway was important shown by migration assay. As well as, CM-2 induced phosphorylation of FAK in tyrosine 397 site but not in CD63 knockouted F3 cells. The present results support that interaction of integrin β1 and PI3K pathway and phosphorylation of FAK are related to induce NSCs migration via CM-2.

Overall, our results reveal that a novel tumor tropism signature of neural stem cells was induced by CM-1 and CM-2. As a result, these chemoattractants improve ability that the inherent tumor-tropism of NSCs to primary and invasive brain tumor foci can be exploited to deliver therapeutics to invasive brain tumor cells in humans. (Imitola et al, 2004) Thus, identification and characterization of such chemoattractants will become to use crucial for brain tumor therapy more efficiently.

For Further studies, we have to establish CM-2 over-expressing NSCs and check whether

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CM-2 is also effective to induce cell migration in vivo. As well as, when CM-1 and CM-2 is introduced in the therapy of actual human brain tumor, we think about that mobility inducers can be used most effectively over a long period of time for therapeutic effect.

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Ⅴ. CONCLUSION

This study showed that both NSCs and CSCs has receptor of CM-1 as integrin αvβ5 and induced migration by CM-1. Moreover, CM-1 enhanced migration ability and elevated tumor targeting for F3.CD. Such F3.CD cells decreased growth rate of tumor as well as prolong survival period in tumor model by converting 5-FC to cytotoxic agents, accordingly it made suicide gene therapy was more effective. Another inducer of NSCs migration, CM-2 induced migration via interaction with integrin β1 and PI3K signaling pathway. The existence of CD63 in NSCs upon interaction with CM-2 activated FAK and then, NSCs induced migration. These results demonstrated that CM-1 and CM-2 as chemoattractants led to migration of NSCs & CSCs and can improve the effect of neural stem cell-based gene therapy for brain tumor.

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CD63 수용체에 의해 세포 이동을 유도하는 것으로 알려져 있다. CM-2에 의한 신경줄기세포의 이동성 유도에 있어서 분자적 메커니즘을 조사하기 위해 migration assay를 수행하였고, 이를 통해 인테그린 β1, PI3K 신호 경로가 CM-2에 의한 신경 줄기 세포 이동의 유도와 연루되어 있다는 것을 확인했다. 또한, CD63 knockouted F3 세포와 pREP4 F3 세포에서 FAK의 타이로신 397자리에 인산화를 확인해본 결과, CD63이 발현되지 않는 신경줄기세포에서 CM-2에 의한 FAK 인산화는 pREP4 F3 세포에 비해 상당히 감소하였다. 종합적으로, 본 내용은 이동성 유도 물질들에 의해 종양-표적화 능력이 향상된 인간 신경 줄기 세포가 독창적인 암 치료법으로 이용될 수 있음을 시사한다.

핵심어 : Neural stem cell, Tumor tropism, Stem cell therapy, Glioblastoma multiforme (GBM), Mobility inducers for neural stem cell