신세포암 세포에서 Ceramide의 성장 억제 효과 및 기전에 관한 연구
Growth Inhibitory Effects and Mechanisms of Ceramides in Renal Cell Carcinoma Cells
소속 상세정보
김청수/Choung Soo Kim
홍준혁/박형근/홍지희/정진수/박노정/황온유/박태한/Jun Hyuk Hong/Hyung Keun Park/Ji Hee Hong/Jin Soo Chung/Ro Jung Park/On You Hwang/Tae Han Park
KMID : 0358319990400030285
Abstract
서론
신세포암이 전이된 경우에는 아직까지 효과적인 치료방법이 없어 그 예후가 아주 불량하
며 5년 생존율이 5% 이하로 보고되고 있다. 근래에 전이된 신세포암에서의 주된 치료방법
은 interferon-α(IFN-α), interleukin-2(IL-2)의 면역요법과 vinblastine 등의 항암요법을 시
행하고 있으나 반응률이 10-30%로 저조하고 interleukin-2와 vinblastine은 각각
capillary-leak syndrome과 neuromuscular toxicity 등의 부작용이 심각하다. 따라서 비교적
효과가 있고 독성이 적은 INF-α의 사용이 주를 이루고 있으나 그 외에 효과가 우수하며
독성이 적은 약물의 개발이 시급한 상태이다.
ceramide는 세포내에서 sphingomyelin cycle의 중간 전달자의 역할을 하여 강력하게 세포
성장을 억제하며 apoptosis를 유발시키는 것으로 보고되고 있다. 이러한 ceramide의 작용을
전이성 신세포암의 치료에 적용하기 위하여 ceramide를 in vitro방법으로 인체의 신세포암
세포에 투여하여 그 효과와 기전을 확인하고 ceramide의 신세포암에 대한 세포독성효과는
apoptosis에 의한 지를 알아보기 위하여 DNA fragmentation assay와 TdT-mediate
biotin-dUTP nicked-end labelling(TUNEL) technique을 시행하였다. 이로써 ceramide에 의
한 세포독성 효과는 apoptosis에 의한 것임을 증명한 후에 athymic mouse에 이식된 인체
신세포암 세포에 대한 ceramide의 종양성장억제 효과를 in vivo 실험에서 비교관찰하여 향
후 전이된 인체의 신세포암에 대한 치료방법으로 ceramide를 이용할 수 있는 실험적 근거
를 수립하고자 하였다.
Purpose : Because metastatic renal cell carcinoma responds to various forms of
therapy with low remission rates, safe therapeutic agents are urgently needed. Ceramide
is a potent and specific suppressor of cell growth and an inducer of apoptosis via an
intracellular mediation of the sphingomyelin cycle. The present was designed to assess
the growth inhibitory effects and their mechanisms of C2-ceramide and C6-ceramide in
renal cell carcinoma cells.
Materials and Methods : A standard microculture tetrazolium(MTT) assay was
measure the cytotoxicity of C2-ceramide and C6-ceramide in renal cell carcimoma cell
line A498. Apoptosis was confirmed by DNA fragmentation assay using agarose gel and
TdT-mediated biotin-dUTP nicked-end labelling(TUNEL) technique. C2-ceramide and
C6-ceramide were injected to the A498 tumor which was formed after A498 cells were
implanted subcutaneously in mice. Growth inhibitory effects of ceramides were examined
biweekly.
Result : The survival fractions of A498 cells were 92.6±0.6, 82.8±14.0, 66.4±11.3, 41.8
±9.6 and 24.3±6.3% for the concentrations of C2-ceramide 2, 4, 6, 8 and 10μM,
repectively. IC50 of C2-ceramide was approximately 6.7μM. The survival
fractions of A498 cell were 60.9±5.0, 23.4±3.0, 8.7±2.1, 5.0±1.2 and 3.3±0.6% for the
concentrations of C6-ceramide 2, 4, 6, 8 and 10μM, respectively. IC50 of
C6-ceramide was about 2.3μM. There were DNA frgmentations in A498 cells treated
with C2-ceramide or C6-ceramide on the gel and apoptotic tumor cells were also
identified after treatment of C2-ceramide and C6-ceramide in TUNEL method. In in vivo
study using athymic mice, the growth of A498 tumors was significantly suppressed by
C2-ceramide and C6-ceramide. In vivo tumor suppressive effect was more ,erst with
C6-ceramide than with C2-ceramide. There's no toxicity-related ceramide-treated athmic
mice for 3 months.
Conclusions : C2-ceramide and C6-ceramide have the growth inhibitory effects human
renal cell carcinoma cell line A498 by apoptosis mechanism in vitro and they have the
in vivo tumor suppressive effects in athymic mice. C6-ceramide was more effective than
C2-ceramide in both in vitro cytotoxicity test and in vivo animal experiment of growth
inhibition. Therefore, ceramides may used to treat metastatic renal cell carcinoma in the
future.
키워드
Renal cell carcinoma; C2-ceramide; C6-ceramide; Cyotoxicity; Apoptosis;
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