[논문]비스테로이드소염제(Nonsteroidal Anti-inflammatory Drug, NSAID)에 의한 인간 암세포의 imatinib 및 TRAIL의 세포 독성 증강 기전 연구

문현정; 강치덕; 김선희

doi:10.5352/jls.2020.30.8.661

[국내논문] 비스테로이드소염제(Nonsteroidal Anti-inflammatory Drug, NSAID)에 의한 인간 암세포의 imatinib 및 TRAIL의 세포 독성 증강 기전 연구
Potentiation of the Cytotoxic Effects of Imatinib and TRAIL by Nonsteroidal Anti-inflammatory Drugs on Human Cancer Cells 원문보기

생명과학회지 = Journal of life science, v.30 no.8, 2020년, pp.661 - 671

문현정 (부산대학교 의학전문대학원 의과학과 생화학교실) , 강치덕 (부산대학교 의학전문대학원 의과학과 생화학교실) , 김선희 (부산대학교 의학전문대학원 의과학과 생화학교실)

초록
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항암 요법의 실패의 주요 원인으로 암세포의 항암제에 대한 내성 획득이 잘 알려져 있다. 비스테로이드소염제(NSAID)는 항염증작용뿐만 아니라 항암제와의 병용요법으로 임상적인 암 치료 요법에 응용되고있다. 본 연구에서는 NSAIDs 인 celecoxib 및 이의 구조 유사체인 2,5-dimethyl celecoxib 그리고 ibuprofen의 인간 암세포에 대한 imatinib 및 TNF-related apoptosis inducing ligand (TRAIL) 세포 독성 변화에 미치는 영향을 조사하였다. NSAID는 TRAIL 및 imatinib에 각각 약제 내성을 나타내는 간암 세포와 백혈병 세포에서 이들 약물의 세포독성을 증강시키는 활성을 나타내었다. NSAID는 ATF4/CHOP의 발현 증강으로 소포체 스트레스 및 오토파지(Autophagy, 자가포식)를 유도하였다. 이로 인한 DR5 발현 증강과 함께 c-FLIP 발현 억제로 TRAIL의 세포독성을 증강시키는 기전을 나타내었다. NSAID로 유도되는 오토파지 활성은 imatinib-resistant CD44^highK562 백혈병세포의 imatinib 감수성을 증강시켰으며, NSAID는 이 세포에서 높은 발현을 나타내는 다양한 stemness-related marker 단백질의 발현 감소를 촉진시키는 활성으로 세포사멸을 유도하는 것을 알 수 있었다. 이러한 결과는 NSAID의 오토파지 유도 활성이 TRAIL과 imatinib의 세포 독성을 증강시키는 것으로서, NSAID와 이들 약물과 병용 처리방법은 인간 암세포의 TRAIL 및 imatinib 내성을 극복 시킴과 동시에 암세포에 이들 약물의 독성 부작용을 감소시킬 수 있는 낮은 농도의 처리를 가능하게 할 것으로 사료된다.

Abstract ▼ AI-Helper

The resistance of cancer cells to anti-cancer drugs is the leading cause of chemotherapy failure. The clinical use of nonsteroidal anti-inflammatory drugs (NSAIDs) has been gradually extended to cancer treatment through combination with anti-cancer drugs. In the current study, we investigated whether NSAIDs including celecoxib (CCB), 2,5-dimethyl celecoxib (DMC), and ibuprofen (IBU) could enhance the cytotoxic effects of imatinib and TNF-related apoptosis inducing ligand (TRAIL) on human cancer cells. We found that the NSAIDs potentiated TRAIL and imatinib cytotoxicity against human hepatocellular carcinoma (HCC) cell lines SNU-354, SNU-423, SNU-449, and SNU-475/TR and against leukemic K562 cells with high level of CD44 (CD44highK562), respectively. More specifically, CCB induced endoplasmic reticulum stress via up-regulation of ATF4/CHOP which is associated with the induction of autophagy against HCC and CD44high K562 cells. NSAID-induced autophagic activity accelerated TRAIL cytotoxicity of HCC cells through up- and down-regulation of DR5 and c-FLIP, respectively. The NSAIDs also potentiated imatinib-induced cytotoxicity and apoptosis through down-regulation of markers in CD44highK562 cells that express a stemness phenotype. Our results suggest that the ability of NSAIDs to induce autophagy could enhance the cytotoxicity of TRAIL and imatinib, leading to a reverse resistance to these drugs in the cancer cells. In conclusion, NSAIDs in combination with low-dose TRAIL or imatinib may constitute a novel clinical strategy that maximizes therapeutic efficacy of each drug and effectively reduces the toxic side effects.

주제어

표/그림 (7)

그림 Fig. 1. Enhancement of TRAIL cytotoxicity by celecoxib or 2, 5-dimethyl celecoxib in HCC and leukemia cells.
그림 Fig. 2. Enhancement of TRAIL cytotoxicity by ibuprofen in various HCC cells.
그림 Fig. 3. Enhanced cell surface expression of DR5 by NSAID in HCC cells.
그림 Fig. 4. Down-regulation of c-FLIP and up-regulation of DR5, and subsequent activation of caspases by NSAID through autophagy induction.
그림 Fig. 5. Effect of NSAID on expression of CHOP/DR5 and c-FLIP through ER-dependent autophagy.
그림 Fig. 6. Potentiation of imatinib-induced cytotoxicity and apoptosis by CCB.
그림 Fig. 7. Acceleration of imatinb-mediated autophagy and down-regulation of stemness-related markers by CCB.

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제안 방법

6A). To evaluate if the combinatorial effect of imatinb and NSAID observed in these cells were correlated with enhancement of imatinib-mediated apoptosis, CD44^highK562 cells were treated with imatinib in combination with either CCB or DMC, which were stained with annexin V/PI, and FACS analysis was performed (Fig. 6B). When compared with CD44^highK562 cells only treated by imatinib, the cells co-treated with imatinib and CCB (or DMC) displayed a significant increase in Annexin V positivity, indicating that NSAID exerted enhanced effects on the induction of im atinib-mediated apoptosis.

대상 데이터

Inhibition of proliferation was evaluated as a percentage of control growth (no drug in the medium). All experiments were carried out in triplicate.
SNU-475/TR cells isolated from parental SNU-475 cells by stepwise increases in concentrations of TRAIL. Human K562 CML cell line was obtained from American Type Culture Collection (Manassas, VA, USA). CD44^highK562 cells were established during isolation of imatinib-resistant K562 cells after treatment with increasing concentrations of imatinib, and were stable in complete medium without imatinib [5].
The antibodies were used: c-FLIP, β-tubulin, ALDH1, Nanog, Oct4, CHOP, DR5, CD44, caspase-8 and caspase-3 (Cell Signaling Technology), poly (ADP-ribose) polymerase (PARP), ATF4 and p53 (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

데이터처리

The statistical significance of differences was assessed using the Student’s t-test.

이론/모형

Cell proliferation was measured by counting viable cells by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric dye-reduction method. Exponentially growing cells (1×10⁴ cells/well) were plated in a 96-well plate and incubated in growth medium treated with the indicated concentrations of TRAIL, imatinib, CCB, DMC or IBU at 37℃.

성능/효과

We also showed that COX-2-independent autophagy inducing effects occurred in cancer cell lines, whether or not they displayed drug-resistant phenotypes. Our results clearly demonstrate that DMC is able to potently mimic CCB with regard to enhancing growth inhibitory of anticancer drugs through induction of autophagy, and thereby DMC and CCB lead to sensitize in drug-resistant HCC and K562 cells to TRAIL and imatinib, respectively. DMC might have an additional advantage: because it does not inhibit COX-2, its use in human patients might not cause the serious cardiovascular side effects that recently emerged with the prolonged use of coxibs such as CCB, rofecoxib, and valdecoxib [21].
4B). To determine whether the ability of CCB to induce autophagy in SNU-449 cells could be linked to the degradation/down-regulation of c-FLIP_L and concurrent up-regulation of DR5 through ER stress-mediated autoph agy, the cells were treated with CCB in the presence of chloroquine (CQ) that blocks an autophagy inhibitor that blocks late stage autophagy, and changed levels of CHOP/ATF4, c-FLIP_L DR5 and cancer stemness-related marker CD44 were evaluated (Fig. 4C). We first determined whether CCB could up-regulate CHOP and ATF4, markers of ER stress.

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