HT-29 monolayer와 spheroid 세포에서의 taxol과 arsenic trioxide에 의한 MAPK의 활성을 확인하였다. 먼저 100 nM taxol을 처리한 경우에는 monolayer와 spheroid 세포에서 모두 ERK1/2가 점차로 활성을 나타내어 30분을 기점으로 그 활성이 감소하는 것으로 나타났다(Figure 9). 그리고 20 μM arsenic trioxide를 처 리한 경우에는 monolayer 세포에서는 10분에서 최대활성을 나타내고 점차 감소하 는 것으로 나타났으며 spheroid 세포에서는 30분에서 최대 활성을 나타내고 점차 로 감소하는 것으로 나타났다(Figure 10). 마지막으로 100 nM taxol과 20 μM arsenic trioxide를 병행처리한 군에서도 역시 monolayer 세포에서는 10분에서 최 대활성을 나타내고 점차 감소하는 것으로 나타났으며 spheroid 세포에서는 30분에 서 최대활성을 나타내고 점차 감소하는 것으로 나타났다(Figure 11). 그리고 모든 처리군에서 p38과 JNK는 활성을 나타내지 않았다(data not shown).
C 10 30 60 120 (mins)
p-MAPK ⇉
MAPK ⇉
Monolayer Spheroid
C 10 30 60 120 (mins)
Figure 9. Immunoblotting analysis of ERK1/2 activation by taxol on the monolayer and the spheroid HT-29 cells. HT-29 cells were treated with 100 nM taxol.
p-MAPK ⇉
MAPK ⇉
Monolayer Spheroid
C 10 30 60 120 (mins)
C 10 30 60 120 (mins)
Figure 10. Immunoblotting analysis of ERK1/2 activation by arsenic trioxide on the monolayer and the spheroid HT-29 cells. HT-29 cells were treated with 20 μM arsenic trioxide.
p-MAPK ⇉
MAPK ⇉
C 10 30 60 120 (mins)
C 10 30 60 120 (mins)
Figure 11. Immunoblotting analysis of ERK1/2 activation by arsenic trioxide and taxol on the monolayer and the spheroid HT-29 cells. HT-29 cells were treated with the combination of 20 μ M arsenic trioxide and 100 nM taxol.
Monolayer Spheroid
제 4 장 고 찰
위하여 collagen-gel 배양, mash-supported organoid 배양(histoculture), multicellular spheroid 배양 등의 3차원적 특성을 나타내는 세포배양 모델이 개발결과 형성된 spheroid 세포는 직경이 1 mm에 이르는 크기의 구상체 형태이었으
HT-29 human colorectal cancer에서 taxol과 arsenic trioxide에 의한 MAP kinase pathway의 활성을 확인한 결과 monolayer 세포와 spheroid 세포 모두에서 ERK1/2의 활성을 관찰할 수 있었다. 그 활성이 10분부터 나타나서 30분에 최대 활성을 나타내다가 점차로 감소하는 성향을 나타내었다. 그러나 p38과 JNK의 경 우는 활성을 나타내지 않았다. 특이하게도 spheroid 세포에서는 control에서도 ERK1/2의 활성이 관찰되었다. 이것은 spheroid 세포를 형성하는 과정에서 영양분 고갈로 인해서 spheroid 세포에 스트레스를 주게되어 ERK1/2를 활성시킨것으로 생각된다.
이상의 결과를 통해서 HT-29 human colorectal cancer의 단층세포에서와 고 형암의 특성에 더욱 근접한 spheroid 세포에서의 동일한 실험의 결과가 많은 부분 에서 차이가 나타남을 알 수 있었다. 즉 in vitro 상태에서 단층세포로 실험한 결 과를 in vivo 상태의 고형암에 직접적으로 적용하는데는 문제가 있음을 알 수 있 었다. 따라서 고형암에 대한 항암제의 효과를 연구하기 위해서는 좀 더 in vivo 상태의 고형암의 특성에 근접한 실험모델이 필요하다는 결론을 얻었고 이 실험모 델로서 spheroid 세포를 사용 할 수 있다는 가능성을 확인하였다.
또 DAPI 분자를 이용한 형광현미경 관찰을 통해 얻어진 결과에서 약제의 고 형암내로의 침투가 용이하지 못함이 약제에 대한 내성을 증가시킨다는 추측을 확 인하는 실험이 앞으로 진행되어져야 할 것이다. 또한 이런 차이가 세포신호전달체 계에서 어떤 차이를 나타내는가를 알아보는 실험도 진행되어야 할 것이다.
제 5 장 결 론
5. Taxol과 arsenic trioxide를 처리한후 10분, 30분, 60분, 120분에서 세포내의 MAP kinase가 활성화되는 것을 관찰한 결과 monolayer 세포와 spheroid 세포에서 모두 ERK1/2가 10분에서부터 활성화 되어 30분을 정점으로 점차 활성이 감소하는 것을 확인하였다.
위의 실험 결과로 볼 때, HT-29 human colorectal cancer cell의 단층세포에 선 taxol과 arsenic trioxide 각각은 세포독성효과를 나타내지만 둘을 병행처리하였 을때는 세포독성효과가 감소함을 확인할 수 있었다. 또한 spheroid 세포를 이용한 실험을 통해서 단층세포에서 나타난 각 약제의 세포독성효과가 현저하게 줄어드 는 것을 확인할 수 있었다. 그리고 DAPI stain을 통한 spheroid 세포의 핵을 염색 한 결과를 통해서 각 약제에 대한 저항성이 약제의 spheroid 세포 내부로의 침투 저해에 의한 것이라는 추측을 할 수 있었다.
이상의 결과를 통해서 고형암의 형태를 가지는 HT-29 human colorectal cancar cell의 경우는 단층세포와 spheroid 세포간에 세포독성효과가 크게 차이가 있음을 확인하였고, arsenic trioxide가 taxol의 효과에 길항작용을 나타냄을 알 수 있었다.
참 고 문 헌
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Abstract
arsenic trioxide decreased the cytotoxicity on the HT-29 monolayer cells. The spheroid cells represented higher resistance against drugs than the monolayer cells. The DNA fragmentations were appeared after incubation with concentrations more than 10 μM arsenic trioxide and 100 nM taxol for 48 h on the monolayer cells. But the results of HT-29 cell line treated with the combination of taxol and arsenic trioxide were disappeared. And we couldn't observe DNA fragmentation in the case of spheroid cells.
These results suggest that apoptosis was not induced in the combinational treatment, which can be thought that arsenic trioxide might work as an antagonist for a taxol mechanism. And the spheroid cells represented higher resistance against drugs than the monolayer cells.
---Key word : taxol, arsenic trioxide, apoptosis, HT-29 cell line, monolayer, spheroid, antagonism