PEGylated TNF-related apoptosis-inducing ligand (TRAIL)-loaded sustained release PLGA microspheres for enhanced stability and antitumor activity

Kim, T.H.,Jiang, H.H.,Park, C.W.,Youn, Y.S.,Lee, S.,Chen, X.,Lee, K.C. Elsevier Science Publishers 2011 Journal of controlled release Vol.150 No.1

The purpose of this work was to develop an effective PEGylated TNF-related apoptosis-inducing ligand (PEG-TRAIL) delivery system for antitumor therapy based on local injection to tumor sites that has a sustained effect without protein aggregation or an initial release burst. The authors designed poly (lactic-co-glycolic) acid (PLGA) microspheres that deliver PEG-TRAIL locally and continuously at tumor sites with sustained biological activity and compared its performance with that of TRAIL microspheres. TRAIL or PEG-TRAIL was microencapsulated into PLGA microspheres using a double-emulsion solvent extraction method. Prepared TRAIL and PEG-TRAIL microspheres showed entirely spherical, smooth surfaces. However, PEG-TRAIL microspheres exhibited a 2.07-fold higher encapsulation efficiency than TRAIL microspheres, and exhibited a tri-phasic in vitro release profile with a lower initial burst (15.8%) than TRAIL microspheres (42.7%). Furthermore, released PEG-TRAIL showed a continued ability to induce apoptosis over 14days. In vivo pharmacokinetic studies also demonstrated that PEG-TRAIL microspheres had a sustained release profile (18days), and that the steady-state concentration of PEG-TRAIL in rat plasma was reached at day 3 and maintained until day 15; its steady-state concentration in rat plasma changed from 1444.3+/-338.4 to 2697.7+/-419.7pg/ml. However, TRAIL microspheres were released out within 2days after administration. Finally, in vivo antitumor tests revealed that tumor growths were significantly more inhibited by a single dose of PEG-TRAIL microspheres than TRAIL microspheres when delivered at 300μg of TRAIL/mouse. Tumors taken from mouse treated with PEG-TRAIL microspheres showed 78.3% tumor suppression at 24days, and this was 3.02-fold higher than that observed for TRAIL microspheres (25.9% tumor inhibition). Furthermore, these improved pharmaceutical characteristics of PEG-TRAIL microspheres resulted in superior therapeutic effects without detectable side effects. These findings strongly suggest that PEG-TRAIL microspheres offer a new therapeutic strategy for the treatment of cancers.

폐암세포주에서 NF-κB 활성 억제를 통한 Proteasome 억제제 MG132의 TRAIL-유도성 Apoptosis 감작 효과

서필원 ( Pil Won Seo ),이계영 ( Kye Young Lee ) 대한결핵 및 호흡기학회 2008 Tuberculosis and Respiratory Diseases Vol.65 No.6

연구배경: 정상세포는 보호되고 종양세포에 독성을 보인다고 알려진 TNF유전자족으로 새로이 확인된 TRAIL이 폐암세포에서 보이는 아포프토시스 효과를 확인하고, 아포프토시스로부터 세포를 보호하는 전사인자 NF-κB가 TRAIL에 의하여 활성화 되는 정도를 평가하여 MG132의 NF-κB활성억제가 TRAIL 유도성 아포프토시스를 감작시키는지를 확인하기 위하여 본 연구를 시행하였다. 방법: A549(wt p53) 및 NCI-H1299(null p53) 폐암세포주를 사용하였다. 세포독성 검사는 MTT assay를 이용하였고 아포프토시스는 Annexin V assay와 FACS 분석을 이용하였다. NF-κB 전사활성은 luciferase reporter gene assay를 이용하였고 IκBα 분해는 western blot을 이용하였으며, TRAIL에 의해 활성화된 NF-κB와 DNA 결합은 electromobility shift assay와 anti-p65 antibody를 이용한 supershift assay로 확인하였다. 결과: 1) TRAIL 100 ng/ml 농도에서 wild-type p53인 A549 폐암세포는 34.4%, p53 null인 NCI-H1299 폐암세포는 26.4%의 세포사를 관찰하였다. 2) Luciferase reporter gene assay로서 TRAIL에 의한 NF-κB의 활성이 A549 IgGκB-luc세포에서 2.45배 증가하고 NCI-H1299 IgGκB-luc세포에서는 1.47배 증가함을 관찰하여 TRAIL에 의하여 NF-κB가 활성화됨을 확인하였다. 3) MG132의 전처치로 TRAIL에 의한 NF-κB의 활성이 A549 세포와 NCI-H1299 세포에서 각각 기저수준의 0.24, 0.21배로 강력히 억제되었다. 4) TRAIL단독으로 30% 전후의 세포독성이 MG132 전처치 후 TRAIL을 투여하면 두 세포주 모두에서 80% 이상의 세포독성이 관찰되어 MG132가 TRAIL유도성 아포프토시스에 감작효과가 있음을 확인하였다. 결론: 이상의 결과로 TRAIL에 상대적인 내성을 보이는 폐암세포주에서 MG132가 NF-κB 활성억제로서 TRAIL유도성 아포프토시스를 강화시키는 효과가 있음을 확인할 수 있었다. 따라서 본 연구는 향후 폐암치료에 있어서 TRAIL유도성 아포프토시스가 이용될 수 있는 가능성을 확인한 기초자료가 된다고 생각된다. Background: TRAIL (TNF-related apoptosis inducing ligand) is a newly identified member of the TNF gene family which appears to have tumor-selective cytotoxicity due to the distinct decoy receptor system. TRAIL has direct access to caspase machinery and induces apoptosis regardless of p53 phenotype. Therefore, TRAIL has a therapeutic potential in lung cancer which frequently harbors p53 mutation in more than 50% of cases. However, it was shown that TRAIL also could activates NF-κB in some cell lines which might inhibit TRAIL-induced apoptosis. This study was designed to investigate whether TRAIL can activate NF-κB in lung cancer cell lines relatively resistant to TRAIL-induced apoptosis and inhibition of NF-κB activation using proteasome inhibitor MG132 which blocks IκBα degradation can sensitize lung cancer cells to TRAIL-induced apoptosis. Methods: A549 (wt p53) and NCI-H1299 (null p53) lung cancer cells were used and cell viability test was done by MTT assay. Apoptosis was confirmed with Annexin V assay followed by FACS analysis. To study NF-κB-dependent transcriptional activation, a luciferase reporter gene assay was used after making A549 and NCI-H1299 cells stably transfected with IgGκ-NF-κB luciferase construct. To investigate DNA binding of NF-κB activated by TRAIL, electromobility shift assay was used and supershift assay was done using anti-p65 antibody. Western blot was done for the study of IκBα degradation. Results: A549 and NCI-H1299 cells were relatively resistant to TRAIL-induced apoptosis showing only 20~30% cell death even at the concentration 100 ng/ml, but MG132 (3μM) pre-treatment 1 hour prior to TRAIL addition greatly increased cell death more than 80%. Luciferase assay showed TRAIL-induced NF-κB transcriptional activity in both cell lines. Electromobility shift assay demonstrated DNA binding complex of NF-κB activated by TRAIL and supershift with p65 antibody. IκBα degradation was proven by western blot. MG132 completely blocked both TRAIL-induced NF-κB dependent luciferase activity and DNA binding of NF-κB. Conclusion: This results suggest that inhibition of NF-κB can be a potentially useful strategy to enhance TRAIL-induced tumor cell killing in lung cancer. (Tuberc Respir Dis 2008;65:476-486)

Ad-TRAIL Induces Apoptosis in Chondrocytes in Vitro and Neutralizing Antibody to TRAIL Prevents The Induction of Apoptosis in Vitro and in Vivo

박진철(Jin Cheol Park),김경택(Kyung Taek Kim),이성원(Sung Won Lee),정원태(Won Tae Chung),유영현(Young Hyun Yoo) 대한해부학회 2005 Anatomy & Cell Biology Vol.38 No.1

본 연구는 관절연골 파괴에 관여하는 것으로 알려진 TRAIL이 골관절염(OA)의 병인에 관여하는지 확인하기 위하여 수행되었다. Primary cultured rat chondrocyte에서 TRAIL과 관련 수용체들의 발현 변화를 관찰하였다. 또 화학적으로 유도된 생쥐 OA 모델 및 외과적으로 유도된 생쥐 골관절염 OA 모델에서의 TRAIL과 Ad-TRAIL을 감염시킨 후 TRAIL과 관련 수용체들의 발현 변화도 관찰하였다. 다음으로 anti-TRAIL neutralizing antibody 로 in vitro 및 in vivo blocking experiment를 실시하였다. Primary cultured rat chondrocyte에서 Ad-TRAIL은 TRAIL 발현을 증가시키고, DR4 발현은 증가시키고 DR5 발현은 감소시켰으며, DcR1 발현은 감소시키고 DcR2 발현에는 영향을 미치지 못하였다. 두 가지 in vivo OA model에서도 OA 연골은 대조군 연골에 비하여 TRAIL과 DR4 발현이 증가하고, DcR1 발현이 감소하며 DcR2 발현에는 변화가 없었다. 그러나 화학적으로 유도된 OA model 에서는 DR5 발현이 줄어들었으나 외과적으로 유도된 OA model에서는 DR5 발현이 다소 증가하였다. In vitro 및 in vivo blocking experiment 모두에서 anti-TRAIL neutralizing antibody는 관절연골세포의 사망을 줄였다. 본 연구결과는 TRAIL이 골관절염의 병인에 관련한다는 것을 증명한다. We undertook this study to investigate whether tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) implicates in the pathogenesis of osteoarthritis (OA). Expression of TRAIL, and death and decoy receptors was examined on the primary cultured chondrocytes and the cartilage obtained from experimentally-induced OA rat model by immunohistochemistry. In vitro and in vivo blocking experiments with anti-TRAIL neutralizing antibody were conducted, and the prevention of cell death was determined by TUNEL assay. Ad-TRAIL infection induced expression of TRAIL and increased expression of death receptor DR4, and decreased expression of DR5 and DcR1 in primary cultured rat chondrocytes. Ad-TRAIL, at 10 and 100 MOI doses, decreased viability of chondrocytes on 4 days after infection. The cartilage obtained from chemically induced OA rat model showed the increased expression of TRAIL and DR4, and the decreased expression of DR5 and DcR1. Whereas surgically induced OA cartilage in vivo showed the increased expression of TRAIL, and DR4 & 5 and the decreased expression of DcR1, compared to the control cartilage. In vitro blocking experiment prevented partially Ad-TRAIL induced death of chondrocytes. Furthermore, in vivo blocking experiment also partially prevented apoptosis in two in vivo OA models. In conclusion, TRAIL-induced chondrocyte apoptosis may play a role in pathogenesis of osteoarthritis.

트레일 사용자의 인구특성과 코로나19 이후 트레일 이용과 트레일 정보 획득에 관한 연구: 외씨버선길과 지리산둘레길 비교분석

권오상 한국거버넌스학회 2022 한국거버넌스학회보 Vol.29 No.2

This paper analyze population characteristics and income of trail users of the Jirisan Trail and Beosun Trail in Korea and compare population characteristics variables with variables of outdoor activities after Covid19, how to get trail information and distance walked. In population characteristics the Jirisan Trail hikers are from Seoul/Gyonggi-do regions, more women than men, high school graduates than college graduates and low income or high income level people. The Beosun Trail hikers are from Seoul and vicinity areas and with more college graduates than high school graduates and more men than women and more over 60 years old comparing the Jirisan Trail. After Covid19, in physical activities ages of 60s or over have walked more but they reduced outdoor activities most too. In outdoor activities after Covid19 walking is 1 st ranked and hiking mountains and bicycle rides are 2 nd and 3 rd ranked in two trails. In hiking distance the Beosun trail men visitors walked 20km per hiking and women walked 10km a visit. However users of the Jirisan walk about 10km regardless of sex. In perceiving the trails hikers of the Jirisan Trail are through travel agencies or hiking organizations and users of the Beosun Trail are through the trail website and guiding posts located in four county regions. To get local information for accommodations and meals the Jirisan Trail users prefer internet search and 2 nd ranked are trail website for men and friends mention for ladies. For the Beosun Trail users get local information by internet web search most and the trail website and info centers are 2 nd ranked. The purpose of visit for two trails are for health 1st and rest 2 nd ranked for both trails.

폐암세포주에서 NFκ 활성 억제를 통한 Proteasome 억제제 MG132의 TRAIL-유도성 Apoptosis 감작 효과

서필원,이계영,Seo, Pil Won,Lee, Kye Young 대한결핵및호흡기학회 2008 Tuberculosis and Respiratory Diseases Vol.65 No.6

연구배경: 정상세포는 보호되고 종양세포에 독성을 보인다고 알려진 TNF유전자족으로 새로이 확인된 TRAIL이 폐암세포에서 보이는 아포프토시스 효과를 확인하고, 아포프토시스로부터 세포를 보호하는 전사인자 $NF-{\kappa}B$가 TRAIL에 의하여 활성화 되는 정도를 평가하여 MG132의 $NF-{\kappa}B$활성억제가 TRAIL 유도성 아포프토시스를 감작시키는지를 확인하기 위하여 본 연구를 시행하였다. 방법: A549(wt p53) 및 NCI-H1299(null p53) 폐암세포주를 사용하였다. 세포독성 검사는 MTT assay를 이용하였고 아포프토시스는 Annexin V assay와 FACS 분석을 이용하였다. $NF-{\kappa}B$ 전사활성은 luciferase reporter gene assay를 이용하였고 $I{\kappa}B{\alpha}$ 분해는 western blot을 이용하였으며, TRAIL에 의해 활성화된 $NF-{\kappa}B$와 DNA 결합은 electromobility shift assay와 anti-p65 antibody를 이용한 supershift assay로 확인하였다. 결과: 1) TRAIL 100 ng/ml 농도에서 wild-type p53인 A549 폐암세포는 34.4%, p53 null인 NCI-H1299 폐암세포는 26.4%의 세포사를 관찰하였다. 2) Luciferase reporter gene assay로서 TRAIL에 의한 $NF-{\kappa}B$의 활성이 A549 $IgG{\kappa}B-luc$세포에서 2.45배 증가하고 NCI-H1299 $IgG{\kappa}B-luc$세포에서는 1.47배 증가함을 관찰하여 TRAIL에 의하여 $NF-{\kappa}B$가 활성화됨을 확인하였다. 3) MG132의 전처치로 TRAIL에 의한 $NF-{\kappa}B$의 활성이 A549 세포와 NCI-H1299 세포에서 각각 기저수준의 0.24, 0.21배로 강력히 억제되었다. 4) TRAIL단독으로 30% 전후의 세포독성이 MG132 전처치 후 TRAIL을 투여하면 두 세포주 모두에서 80% 이상의 세포독성이 관찰되어 MG132가 TRAIL유도성 아포프토시스에 감작효과가 있음을 확인하였다. 결론: 이상의 결과로 TRAIL에 상대적인 내성을 보이는 폐암세포주에서 MG132가 $NF-{\kappa}B$ 활성억제로서 TRAIL유도성 아포프토시스를 강화시키는 효과가 있음을 확인할 수 있었다. 따라서 본 연구는 향후 폐암치료에 있어서 TRAIL유도성 아포프토시스가 이용될 수 있는 가능성을 확인한 기초자료가 된다고 생각된다. Background: TRAIL (TNF-related apoptosis inducing ligand) is a newly identified member of the TNF gene family which appears to have tumor-selective cytotoxicity due to the distinct decoy receptor system. TRAIL has direct access to caspase machinery and induces apoptosis regardless of p53 phenotype. Therefore, TRAIL has a therapeutic potential in lung cancer which frequently harbors p53 mutation in more than 50% of cases. However, it was shown that TRAIL also could activates $NF-{\kappa}B$ in some cell lines which might inhibit TRAIL-induced apoptosis. This study was designed to investigate whether TRAIL can activate $NF-{\kappa}B$ in lung cancer cell lines relatively resistant to TRAIL-induced apoptosis and inhibition of $NF-{\kappa}B$ activation using proteasome inhibitor MG132 which blocks $I{\kappa}B{\alpha}$ degradation can sensitize lung cancer cells to TRAIL-induced apoptosis. Methods: A549 (wt p53) and NCI-H1299 (null p53) lung cancer cells were used and cell viability test was done by MTT assay. Apoptosis was confirmed with Annexin V assay followed by FACS analysis. To study $NF-{\kappa}B$-dependent transcriptional activation, a luciferase reporter gene assay was used after making A549 and NCI-H1299 cells stably transfected with IgG ${\kappa}-NF-{\kappa}B$ luciferase construct. To investigate DNA binding of $NF-{\kappa}B$ activated by TRAIL, electromobility shift assay was used and supershift assay was done using anti-p65 antibody. Western blot was done for the study of $I{\kappa}B{\alpha}$ degradation. Results: A549 and NCI-H1299 cells were relatively resistant to TRAIL-induced apoptosis showing only 20~30% cell death even at the concentration 100 ng/ml, but MG132 ($3{\mu}M$) pre-treatment 1 hour prior to TRAIL addition greatly increased cell death more than 80%. Luciferase assay showed TRAIL-induced $NF-{\kappa}B$ transcriptional activity in both cell lines. Electromobility shift assay demonstrated DNA binding complex of $NF-{\kappa}B$ activated by TRAIL and supershift with p65 antibody. $I{\kappa}B{\alpha}$ degradation was proven by western blot. MG132 completely blocked both TRAIL-induced $NF-{\kappa}B$ dependent luciferase activity and DNA binding of $NF-{\kappa}B$. Conclusion: This results suggest that inhibition of $NF-{\kappa}B$ can be a potentially useful strategy to enhance TRAIL-induced tumor cell killing in lung cancer.

TRAIL 유도 세포사멸에 있어서 IFN-γ의한 증가 기전 연구: IRF-1과의 관련성

박상열,설재원,이유진,강석진,김인식,강형섭,채준석,조종후,Park, Sang-Youel,Seol, Jae-Won,Lee, You-Jin,Kang, Seog-Jin,Kim, In-shik,Kang, Hyung-sub,Chae, Joon-seok,Cho, Jong-Hoo 대한수의학회 2004 大韓獸醫學會誌 Vol.44 No.2

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family and potent inducer of apoptosis. TRAIL has been shown to effectively limit tumor growth in vivo without detectable cytotoxic side effects. Interferon (IFN)-${\gamma}$ often modulates the anti-cancer activities of TNF family members including TRAIL. We previously reported that IFN-${\gamma}$ enhanced TRAIL-induced Apoptosis in HeLa cells without the unknown mechanism. In this study, we investigated whether IRF-1 involves in IFN-${\gamma}$-enhanced TRAIL-induced apoptosis. We exposed HeLa cells to IFN-${\gamma}$ for 12 hours and then treated with recombinant TRAIL protein. No apoptosis was induced in cells pretreated with IFN-${\gamma}$, and TRAIL only induced 30% apoptosis after 3 hours treatment. In HeLa cells pretreated with IFN-${\gamma}$, TRAIL induced cell death to more than 75% at 3 hours, showed that IFN-${\gamma}$-pretreatment enhanced HeLa cell death to TRAIL-induced apoptosis. To investigate the functional role of IRF-1 in IFN-${\gamma}$-enhanced TRAIL-induced apoptosis, IRF-1 was overexpressed by using an adenoviral vector AdIRF-1. IRF-1 overexpression increased apoptotic cell death and significantly enhanced apoptotic cell death induced by TRAIL when infected cells were treated with TRAIL. Our findings show that IFN-${\gamma}$ enhances TRAIL-induced apoptosis by IRF-1 in HeLa cells.

인체 방광암세포에서 histone deacetylase 억제제인 sodium butyrate이 TRAIL에 의한 apoptosis 유도에 미치는 영향

한민호(Min-Ho Han),최영현(Yung Hyun Choi) 한국생명과학회 2016 생명과학회지 Vol.26 No.4

TRAIL은 정상세포에서는 세포독성을 나타내지 않는 반면, 암세포에서는 사멸을 유도하므로 항암제로 각광받고 있지만 많은 암세포에서 TRAIL에 저항성을 가지고 있는 것으로 알려져 있으므로 이를 극복해야하는 큰 어려움이 남아있다. 본 연구에서는 TRAIL에 저항성을 가지는 인간 방광암 세포주인 5637 세포를 이용하여 histone deacetylase 억제제인 sodium butyrate (SB)와 TRAIL을 혼합처리하였을 경우 유발되는 세포사멸 효과와 이와 관련된 분자생물학적 메카니즘을 연구하였다. 세포독성이 없는 조건의 TRAIL과 SB를 혼합처리 하였을 경우 SB 단독처리군 보다 세포사멸이 현저하게 증가하는 것으로 확인되었다. TRAIL과 SB의 혼합처리는 caspases (caspase-3, -8 and -9)의 활성화 및 PARP의 단편화를 유발하였다. 하지만 caspase 억제제에 의하여 TRAIL과 SB의 혼합처리에 의하여 유발되는 apoptosis가 현저하게 억제되는 것으로 나타났다. 또한 TRAIL과 SB의 혼합처리는 세포표면에 존재하는 DR5의 발현 증가 및 c-FLIP의 발현 감소를 유발하였으며, pro-apoptotic protein인 Bax와 세포질cytochrome c의 발현 증가 및 anti- apoptotic protein인 Bcl-xL의 발현감소와 함께 tBid의 형성을 유발하였다. 이는 SB와 TRAIL의 혼합처리가 안전하고 선택적으로 TRAIL에 저항성을 가지는 방광암 세포에서 치료하는데 효과적인 전략임을 제시하는 결과이다. The tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is considered a promising anticancer agent due to its unique ability to induce cancer cell death having only negligible effects on normal cells. However, many cancer cells tend to be resistant to TRAIL. In this study, we investigated the effects and molecular mechanisms of sodium butyrate (SB), a histone deacetylase inhibitor, in sensitizing TRAIL-induced apoptosis in 5637 human bladder cancer cells. Our results indicated that co-treatment with SB and TRAIL significantly increased the apoptosis induction, compared with treatment with either agent alone. Co-treatment with SB and TRAIL effectively increased the cell-surface expression of death receptor (DR) 5, but not DR4, which was associated with the inhibition of cellular Fas-associated death domain (FADD)-like interleukin-1β-converting enzyme (FLICE) inhibitory protein (c-FLIP). Furthermore, the activation of caspases (caspase-3, -8 and -9) and degradation of poly(ADPribose) were markedly increased in 5637 cells co-treated with SB and TRAIL; however, the synergistic effect was perfectly attenuated by caspase inhibitors. We also found that combined treatment with SB and TRAIL effectively induced the expression of pro-apoptotic Bax, cytosolic cytochrome c and cleave Bid to truncated Bid (tBid), along with down-regulation of anti-apoptotic Bcl-xL expression. These results collectively suggest that a combined regimen of SB plus TRAIL may offer an effective therapeutic strategy for safely and selectively treating TRAIL-resistant bladder cancer cells.

갑상선암 세포주에서 Troglitazone에 의한 TRAIL-유도 세포소멸 감수성의 증가

박진우,OrloH.Clark<SUP>1<.SUP>.Jin-Woo Park and Orlo H. Clark<SUP>1<.SUP> 대한갑상선-내분비외과학회 2003 The Koreran journal of Endocrine Surgery Vol.3 No.2

Purpose: Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) induces apoptosis in many human cancer cells but not in normal cells. Thyroid cancer cells, however, appear to be relatively resistant to TRAIL-induced apoptosis. We investigated the effect of troglitazone, a PPARγ agonist, on TRAIL-induced apoptosis in thyroid cancer cells. Methods: We used 6 thyroid cancer cell lines: TPC-1, FTC- 133, FTC-236, FTC-238, XTC-1, and ARO82-1. We used flow cytometry to detect apoptosis and used MTT assay to measure anti-proliferation effects. ANOVA was used for statistical analysis. Results: TPC-1 cells were the most sensitive to soluble TRAIL. FTC-133 and ARO82-1 were resistant to TRAIL and growth inhibition was less than 20% at concentration of 800 ng/ml of TRAIL. In both TPC-1 (TRAIL-sensitive) and FTC- 133 (TRAIL-resistant) thyroid cancer cell lines, pretreatment with troglitazone enhanced TRAIL-induced cell death significantly. Bcl-family proteins did not seem to be involved in sensitization of TRAIL-induced apoptosis by troglitazone. Conclusion: TRAIL in combination with troglitazone induces apoptosis in thyroid cancer cells at suboptimal concentrations that can not be achieved using TRAIL alone. (Korean J Endocrine Surg 2003;3:113-120)

폐암세포주에서 IGF-1R 억제를 이용한 TRAIL 및 gefitinib에 대한 감수성 증가를 위한 연구

이윤진 ( Yoon Jin Lee ),박미영 ( Mi Young Park ),강영애 ( Young Ae Kang ),권성연 ( Sung Youn Kwon ),윤호일 ( Ho Il Yoon ),이재호 ( Jae Ho Lee ),이춘택 ( Choon Taek Lee ) 대한결핵 및 호흡기학회 2007 Tuberculosis and Respiratory Diseases Vol.63 No.1

배경: 폐암의 새로운 치료제로 각광을 받고 있는 TRAIL은 암에 선택적으로 apoptosis를 일으킨다고 알려진 cytokine으로 알려져 있다. 또한 gefitinib (Iressa)는 폐암의 적용된 최초의 표적치료제로 각광을 받고 있다. 그러나 일부의 암세포에서는 이에 대한 저항성을 보이고 있다. 본 연구에서는 암세포에서 외부의 apoptotic 자극에 저항성을 보이는 IGF-1R를 억제함으로 TRAIL 및 gefitinib의 항암작용을 증가시키고자 실험을 시행하였다. 방법: 암세포주는 TRAIL 및 gefitinib에 민감한 NCI H460와 두 약제에 중등도의 저항성을 보이는 A549의 폐암세포주로 시행하였으며 IGF-1R의 억제는 본 연구자가 개발하였던 IGF-1 pathway를 dominant negative inhibition을 할 수 있는 adenovirus-IGF1R(482 ST, 950ST) 및 IGF-1R tyrosine kinase inhibitor인 Tyrphostin AG1024를 사용하였고 gefitinib에 대한 실험에서는 RNA interference를 이용하여 IGF-1R의 발현을 억제하는 adenovirus- shIGF-1R을 이용하였다. 결과: TRAIL에 저항성을 보이는 폐암세포주(A549)에서 adenovirus-IGF1R(482ST, 950ST) 및 AG1024로 IGF-1R를 억제한 후 TRAIL을 투여한 경우 항암효과가 증대되었다. A549에 adenovirus- shIGF-1R을 감염시켜 IGF-1R의 발현을 억제한 결과 gefitinib에 대한 감수성이 증가되었다. 결론: 여러 기전을 이용한 IGF-1R의 억제는 TRAIL 및 gefitinib에 대해 저항성을 보이던 폐암세포주의 감수성을 향상시킴을 확인하였으며 이는 폐암의 임상치료의 한계를 극복할 수 있는 하나의 단초를 제공하였다. 향후 여러 종류의 암세포주에서의 검증 및 in vivo 실험이 필요하리라 생각된다. Background: TRAIL is a cytokine that selectively induces apoptosis in various cancer cell lines. Gefitinib is new targeted drug applied in lung cancer that selectively inhibits EGFR tyrosine kinase. However, lung cancers have shown an initial or acquired resistance to these drugs. This study examined the effect of IGF-1R and its blockade on enhancing the sensitivity of lung cancer cell lines to TRAIL and gefitinib. Methods: Two lung cancer cell lines were used in this study. NCI H460 is very sensitive to TRAIL and gefitinib. On the other hand, A549 shows moderate resistance to TRAIL and gefitinib. The IGF-1R blockade was performed using adenoviruses expressing the dominant negative IGF-1R and shRNA to IGF-1R and AG1024 (IGF-1R tyrosine kinase inhibitor). Results: The adenovirus expressing dominant negative IGF-1R(950st) induced the increased expression of defective IGF-1R on the lung cancer cell surface, and the adenovirus-shIGF-1R effectively decreased the level of IGF-1R expression on cell surface. The genetic blockade of IGF-1R by the adenovirus-dnIGF-1R and AG1024 increased the sensitivity of A549 cells to TRAIL. The reduction of IGF-1R by transduction with ad-shIGF-1R also increased the sensitivity of the A549 cells to gefitinib. Conclusion: The blockade of IGF-1R through various mechanisms increased the sensitivity of the lung cancer cell line that was resistant to TRAIL and gefitinib. However, further studies using other cell lines showing acquired resistance as well as in vivo animal experiments will be needed. (Tuberc Respir Dis 2007; 63: 42-51)

Quercetin Potentiates TRAIL-induced Apoptosis in Human Colon KM12 Cells

Jun-Ik Park(박준익),Hak-Bong Kim(김학봉),Mi-Ju Kim(김미주),Jae-Won Lee(이재원),Jae-Ho Bae(배재호),Soo-Jung Park(박수정),Dong-Wan Kim(김동완),Chi-Dug Kang(강치덕),Sun-Hee Kim(김선희) 한국생명과학회 2009 생명과학회지 Vol.19 No.9

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 KM12 세포도 TRAIL에 내성을 나타낸다. 본 연구에서는 KM12세포의 TRAIL 내성에 대한 새로운 표적 분자의 발굴과 이를 토대로 한 새로운 내성극복 방법을 연구하였다. 새로운 TRAIL sensitizer로서 quercetin 을 발굴하고, 이를 KM12세포에 TRAIL 과 병용 처리하여TRAIL의 효과증강을 시도하였다. KM12세포에서 quercetin은 c-FLIP 의 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 KM12세포의 TRAIL-sensitization에 사용될 수 있음을 제시하였다. 이러한 연구 결과는 대장암의 치료 시 TRAIL과 quercetin 병용하므로서 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 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.