Adeno-associated virus expressing endostatin in combination with topoisomerase inhibitor or HIF-1 inhibitor blocks hypoxia-inducible angiogenesis in a mouse model

Abstract

[한글]

목적: 유전자 치료를 함에 있어 재조합 adeno-asociated virus 매개 endostatin이 암을 치료하는 하나의 새로운 방법으로 조사되고 있다. 그러나 치료효과를 위해서는 지속적으로 많은 양의 단백질이 필요하다. 그리고 topoisomerase inhibitor, hif1 inhibitor, eupatilin등을 재조합 adeno-asociated virus 매개 endostatin과 함께 병용투여 함으로써 유전자치료를 극대화 시키고자 한다. 저산소 상태에서 hif1과 STAT3가 안정적으로 발현하면 VEGF또한 안정적으로 발현한다. 따라서 hif1과 VEGF의 발현을 감소시킴으로 STAT3의 발현을 억제할 수 있기에 STAT3의 발현을 감소 시킬 수 있는 유전자 치료를 하나의 임상 적용에 이용하기 위한 방법을 제시하고자 한다.

방법: 재조합 adeno-asociated virus 매개 endostatin을 간암세포주에 transduction시킨다. topoisomerase inhibitor, hif1 inhibitor, eupatilin등이 endostatin발현에 영향을 끼치는지를 테스트하기위해 Western blotting, Chromatin IP, ELISA등을 수행하였고, endostatin의 생물학적 활성을 endothelial proliferation, endothelial tube formation방법을 사용하였다. 재조합 adeno-asociated virus 매개 endostatin과 약물의 병용투여로 인한 항암효과를 알아보기 위해 동물실험, 조직 면역 화학 염색등을 하였다.

결과 : VEGF promoter상에서 HIF1, STAT3등이 recruitment되고 저산소 상태에서 재조합 adeno-asociated virus를 발현하는 endostatin과 eupatilin이 hif1과 VEGF의 발현을 감소시킴으로 STAT3의 발현을 억제하였다. 이로써 암을 타겟으로 하는 유전자 치료에 있어 재조합 adeno-asociated virus를 발현하는 endostatin만 투여하는 것 보다는 eupatilin과의 병용투여가 훨씬 더 상승효과가 높기에 앞으로 유전자 치료를 실제 임상에 적용함에 있어 STAT3의 발현을 억제하는 새로운 기술 방법이 되기를 기대한다.

[영문]

AIM: rAAV mediated endostatin gene therapy has been examined as a new method for treating cancer. However, a sustained and high protein delivery is required to achieve the desired therapeutic effects. We evaluated the impact of topoisomerase inhibitors or hif1 inhibitors in rAAV delivered endostatin gene therapy in a liver tumor model. We demonstrated that STAT3 is a novel and essential component of HIF-1 regulatory machinery through associating with HIF-1 under hypoxic conditions STAT3 inhibition can be an application for clinical therapy with solid tumors by reducing HIF-1 protein induction and VEGF expression.

METHODS: rAAV containing endostatin expression cassettes were transduced into hepatoma cell lines. To test whether the topoisomerase inhibitor or hif1 inhibitor pretreatment increased the expression of endostatin, Western blotting and ELISA were performed. The biologic activity of endostatin was confirmed by endothelial cell proliferation and tube formation assays.

The anti-tumor effects of the rAAV-endostatin vector combined with hif1 inhibitors, were evaluated in a mouse liver tumor model.

RESULTS: Topoisomerase inhibitors, including camptothecin and etoposide, and hif1 inhibitors were found to increase the endostatin expression level in vitro. The over-expressed endostatin, as a result of pretreatment with a topoisomerase inhibitor or hif1 inhibitors , was also biologically active. In animal experiments, the combined therapy of hif1 inhibitor with the rAAV-endostatin vector had the best tumorsuppressive effect and tumor foci were barely observed in livers of the treated mice. Pretreatment with an eupatilin increased the level of endostatin in the liver and serum of rAAV-endostatin treated mice. Finally, the mice treated with rAAV-endostatin in combination with eupatilin showed the longest survival among the experimental models.

CONCLUSION: Our results demonstrate that STAT3 is a new regulator of VEGF expression under hypoxic conditions and hypoxia-mediated VEGF expression requires binding of both STAT3 and HIF-1 to the VEGF promoter for maximum induction in mouse hepatoma cells. Also, rAAV delivered endostatin gene therapy in combination with a topoisomerase inhibitor, a hif1 inhibitor or eupatilin pretreatment is an effective modality for anticancer gene therapy.