많은 암세포에 선택적 세포독성을 나타내는 TNF-related apoptosis-inducing ligand (TRAIL)는 유효한 항암제로서 사용될 수 있지만, TRAIL에 내성을 나타내는 암세포에는 TRAIL-sensitization의 방법이 필요하다. 사람 대장암 세포인 KMl2 세포도 TRAIL에 내성을 나타낸다. 본 연구에서는 KMl2세포의 TRAIL 내성에 대한 새로운 표적 분자의 발굴과 이를 토대로 한 새로운 내성극복 방법을 연구하였다. 새로운 TRAIL sensitizer로서 quercetin을 발굴하고, 이를 KMl2세포에 TRAIL과 병용 처리하여 TRAIL의 효과증강을 시도하였다. KMl2세포에서 quercetin은 c-FLTP의 발현을 감소시키고, DNA-PK/Akt 신호전달경로를 억제하므로서, death receptors (DR4/DR5) 발현을 증강시켰다. 또한 caspases (caspase 3, -8 및 -9)활성 증강과 PARP cleavage, 이에 따른 Bax의 발현을 증강시키는 기전으로 TRAIL에 의한 apoptosis를 증대시키는 활성이 있음을 밝혔다. 즉 quercetin이 KMl2세포의 TRAIL-sensitization에 사용될 수 있음을 제시하였다. 이러한 연구 결과는 대장암의 치료 시 TRAIL과 quercetin병용하므로서 치료 효과를 높일 수 있는 새로운 약제 병용 방법을 제시하였고, 다른 TRAIL 내성 종양에 응용 될 수 있음을 시사하였다.
많은 암세포에 선택적 세포독성을 나타내는 TNF-related apoptosis-inducing ligand (TRAIL)는 유효한 항암제로서 사용될 수 있지만, TRAIL에 내성을 나타내는 암세포에는 TRAIL-sensitization의 방법이 필요하다. 사람 대장암 세포인 KMl2 세포도 TRAIL에 내성을 나타낸다. 본 연구에서는 KMl2세포의 TRAIL 내성에 대한 새로운 표적 분자의 발굴과 이를 토대로 한 새로운 내성극복 방법을 연구하였다. 새로운 TRAIL sensitizer로서 quercetin을 발굴하고, 이를 KMl2세포에 TRAIL과 병용 처리하여 TRAIL의 효과증강을 시도하였다. KMl2세포에서 quercetin은 c-FLTP의 발현을 감소시키고, DNA-PK/Akt 신호전달경로를 억제하므로서, death receptors (DR4/DR5) 발현을 증강시켰다. 또한 caspases (caspase 3, -8 및 -9)활성 증강과 PARP cleavage, 이에 따른 Bax의 발현을 증강시키는 기전으로 TRAIL에 의한 apoptosis를 증대시키는 활성이 있음을 밝혔다. 즉 quercetin이 KMl2세포의 TRAIL-sensitization에 사용될 수 있음을 제시하였다. 이러한 연구 결과는 대장암의 치료 시 TRAIL과 quercetin병용하므로서 치료 효과를 높일 수 있는 새로운 약제 병용 방법을 제시하였고, 다른 TRAIL 내성 종양에 응용 될 수 있음을 시사하였다.
Many cancer cells are sensitive to the TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. However, some cancer cells show either partial or complete resistance to TRAIL. Human colon carcinoma KM12 cells have been shown to be insensitive to TRAIL-induced apoptosis. To overcome TRAIL res...
Many cancer cells are sensitive to the TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. However, some cancer cells show either partial or complete resistance to TRAIL. Human colon carcinoma KM12 cells have been shown to be insensitive to TRAIL-induced apoptosis. To overcome TRAIL resistance in KM12 cells, we targeted key anti-apoptotic molecules involved in the modulation of TRAIL resistance in the cells, and evaluated the effects of quercetin as a TRAIL sensitizer in the cells. We found that quercetin acted in synergy with TRAIL to enhance TRAIL-induced apoptosis in KM12 cells by the down-regulation of c-FLIP and DNA-PKcs/Akt and up-regulation of death receptors (DR4/DR5), which led to the enhancement of TRAIL-mediated activation of caspases and subsequent cleavage of PARP, as well as up-regulation of Bax. These findings suggest that the DNA-PKcs/Akt signaling pathway, as well as c-FLIP, play essential roles in regulating cells in the escape from TRAIL-induced apoptosis. Based on these results, this study provides a potential application of quercetin in combination with TRAIL in the treatment of human colon cancer.
Many cancer cells are sensitive to the TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. However, some cancer cells show either partial or complete resistance to TRAIL. Human colon carcinoma KM12 cells have been shown to be insensitive to TRAIL-induced apoptosis. To overcome TRAIL resistance in KM12 cells, we targeted key anti-apoptotic molecules involved in the modulation of TRAIL resistance in the cells, and evaluated the effects of quercetin as a TRAIL sensitizer in the cells. We found that quercetin acted in synergy with TRAIL to enhance TRAIL-induced apoptosis in KM12 cells by the down-regulation of c-FLIP and DNA-PKcs/Akt and up-regulation of death receptors (DR4/DR5), which led to the enhancement of TRAIL-mediated activation of caspases and subsequent cleavage of PARP, as well as up-regulation of Bax. These findings suggest that the DNA-PKcs/Akt signaling pathway, as well as c-FLIP, play essential roles in regulating cells in the escape from TRAIL-induced apoptosis. Based on these results, this study provides a potential application of quercetin in combination with TRAIL in the treatment of human colon cancer.
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가설 설정
In search of an effective treatment strategy for TRAIL-re- sistant KM12 cellsz we evaluated whether polyphenol quer- cetin could act as a potent sensitizer of TRAIL in the KM12 cells. When KM12 cells were treated with TRAIL (1- to 50 ng/ ml) in the absence or presence of quercetin (10- or 100 uM) for 96 hrz the susceptibility to TRAIL-induced cytotox- icity was measured by the MTT assay.
제안 방법
In this study, we found that quercetin acted synergisti- cally with TRAIL to induce apoptosis in human colon carci- noma KM12 cellsz and it occurs through new mechanism of the potentiation by quercetin of TRAIL. We revealed that the down-regulation of c-FLIP and DNA-PKcs/Akt signal- ing pathway by quercetin treatment which overcame the TRAIL-resistance in the KM12 cells.
대상 데이터
The human colon carcinoma cell line KM12 was provided by Dr. Isaih J. Fidler (University of Texas M.D. Anderson Cancer Center, Houston, TXZ USA). The KM12 cells were maintained in culture (5% CO2 and 95% air at 37℃) as adherent monolayers in DMEM supplemented with 10% fetal bovine serum, sodium pyruvate, nonessential amino acids, L-glutamine, and vitamin solution.
데이터처리
of at least three independent experiments. The statistical sig- nificance of differences assessed using the Student's t-test and two-way ANOVA (GraphPad software; GraphPad, Santiago, CA) with Bonferroni posttests. p<0.
이론/모형
Cell proliferation was measured by the 3-(4/ 5-dimethylth- iazol-2-yl)-2/5-diphenyltetrazolium bromide (MTT; Sigma Chemical Company, St. Louis, MO.Z USA) colorimetric dye-reduction method. Exponentially growing cells (IxlO4 cells/well) were plated in a 96 well plate and incubated in growth medium treated under the indicated condition with TRAIL and/or quercetin at 37℃.
When KM12 cells were treated with TRAIL (1- to 50 ng/ ml) in the absence or presence of quercetin (10- or 100 uM) for 96 hrz the susceptibility to TRAIL-induced cytotox- icity was measured by the MTT assay. As shown in Fig.
성능/효과
In conclusion, the finding of this study provides important insights into how quercetin promotes TRAIL-induced apoptosis in TRAIL-resistant KM12 cells. This model provides a framework for overcoming of TRAIL resistance in other cancer cells.
Indeed, it has been reported that Akt may control the cytotoxic effect of TRAIL by modulating c-FLIPs expression in human gastric cancer cells [26]. Our results also showed that sensitization of KM12 cells by quercetin to TRAIL-induced apoptosis might be closely associated with down-regulation of the Akt/c-FLIP pathway.
We demonstrate here that combined treatment with quercetin and TRAIL effectively induces apoptotic cell death in TRAIL-resistant KM12 cells. The results of this study suggest that the down-regulation of c-FLIP and DNA-PKcs/ Akt signaling pathway by treatment with quercetin might be involved in the sensitization of KM12 cells to TRAIL-induced apoptosis.
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