• Molecules and Cells
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• 제25권2호
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• pp.184-195
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• 2008

We examined the role of c-FLIP in the motility of HeLa cells. A small interfering RNA (siRNA) directed against c-FLIP inhibited the adhesion and motility of the cells without affecting their growth rate. The long form of c-FLIP ($c-FLIP_L$), but not the short form ($c-FLIP_S$), enhanced adhesion and motility. Downregulation of $c-FLIP_L$ with siRNA decreased phosphorylation of FAK and ERK, while overexpression of $c-FLIP_L$ increased their phosphorylation. Overexpression of FAK activated ERK, and enhanced the motility of HeLa cells. FRNK, an inhibitory fragment of FAK, inhibited ERK and decreased motility. Inhibition of ERK also significantly suppressed $c-FLIP_L$-promoted motility. Inhibition of ROCK by Y27632 suppressed the $c-FLIP_L$-promoted motility by reducing phosphorylation of FAK and ERK. Overexpression of $c-FLIP_L$ increased the expression and secretion of MMP-9, and inhibition of MMP-9 by Ilomastat reduced $c-FLIP_L$- promoted cell motility. A caspase-like domain (amino acids 222-376) was found to be necessary for the $c-FLIP_L$-promoted cell motility. We conclude that $c-FLIP_L$ promotes the motility of HeLa cells by activating FAK and ERK, and increasing MMP-9 expression.

• Asian-Australasian Journal of Animal Sciences
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• 제23권3호
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• pp.401-409
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• 2010

FLICE inhibitory protein (FLIP) is one of the important anti-apoptotic proteins in the Fas/FasL apoptotic path which has death effect domains, mimicking the pro-domain of procaspase-8. To reveal the intracellular signal transduction molecules involved in the process of follicular development in the bovine ovary, we cloned the c-FLIP(L) gene in bovine ovary tissue with the reverse transcription polymerase chain reaction (RT-PCR), deleted the termination codon in its cDNA, and directionally cloned the amplified c-FLIP(L) gene into eukaryotic expression vector pAcGFP-Nl, including AcGFP, and successfully constructed the fusion protein recombinant plasmid. After identifying by restrictive enzyme BglII/EcoRI and sequencing, pAcGFP-bFLIP(L) was then transfected into follicular granulosa cells, mediated by Lipofectamine 2000, the expression of AcGFP observed and the transcription and expression of c-FLIP(L) detected by RT-PCR and Western blot. The results showed that the cattle c-FLIP(L) was successfully cloned; the pAcGFPbFLIP(L) fusion protein recombinant plasmid was successfuly constructed by introducing a BglII/EcoRI cloning site at the two ends of the c-FLIP(L) open reading frame and inserting a Kozak sequence before the start codon. AcGFP expression was detected as early as 24 h after transfection. The percentage of AcGFP positive cells reached about 65% after 24 h. A 1,483 bp transcription was amplified by RT-PCR, and a 83 kD target protein was detected by Western blot. Construction of the pAcGFP-bFLIP(L) recombinant plasmid should be helpful for further understanding the mechanism of regulation of c-FLIP(L) on bovine oocyte formation and development.

• BMB Reports
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• 제47권9호
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• pp.488-493
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• 2014

Adaptor protein FADD forms the death inducing signaling complex (DISC) by recruiting the initiating caspases-8 and -10 through homotypic death effector domain (DED) interactions. Cellular FLICE-inhibitory protein (c-FLIP) is an inhibitor of death ligand-induced apoptosis downstream of death receptors, and FADD competes with procaspase-8/10 for recruitment for DISC. However, the mechanism of action of FADD and c-FLIP proteins remain poorly understood at the molecular level. In this study, we provide evidence indicating that the death effector domain (DED) of FADD interacts directly with the death effector domain of human c-FLIP. In addition, we use homology modeling to develop a molecular docking model of FADD and c-FLIP proteins. We also find that four structure-based mutants (E80A, L84A, K169A and Y171A) of c-FLIP DEDs disturb the interaction with FADD DED, and that these mutations lower the stability of the c-FLIP DED.

• Biomolecules & Therapeutics
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• 제23권1호
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• pp.31-38
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• 2015

Histone acetylation plays a critical role in the regulation of transcription by altering the structure of chromatin, and it may influence the resistance of some tumor cells to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) by regulating the gene expression of components of the TRAIL signaling pathway. In this study, we investigated the effects and molecular mechanisms of trichostatin A (TSA), a histone deacetylase inhibitor, in sensitizing TRAIL-induced apoptosis in Caki human renal carcinoma cells. Our results indicate that nontoxic concentrations of TSA substantially enhance TRAIL-induced apoptosis compared with treatment with either agent alone. Cotreatment with TSA and TRAIL effectively induced cleavage of Bid and loss of mitochondrial membrane potential (MMP), which was associated with the activation of caspases (-3, -8, and -9) and degradation of poly (ADP-ribose) polymerase (PARP), contributing toward the sensitization to TRAIL. Combined treatment with TSA and TRAIL significantly reduced the levels of the cellular Fas-associated death domain (FADD)-like interleukin-$1{\beta}$-converting enzyme (FLICE) inhibitory protein (c-FLIP), whereas those of death receptor (DR) 4, DR5, and FADD remained unchanged. The synergistic effect of TAS and TRAIL was perfectly attenuated in c-$FLIP_L$-overexpressing Caki cells. Taken together, the present study demonstrates that down-regulation of c-FLIP contributes to TSA-facilitated TRAIL-induced apoptosis, amplifying the death receptor, as well as mitochondria-mediated apoptotic signaling pathways.

• 생명과학회지
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• 제22권10호
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• pp.1277-1285
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• 2012

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)은 암세포 선택적으로 작용하므로서 유용한 항암제로 주목 받고 있다. 그러나, TRAIL 에 내성을 나타내는 암세포도 많이 존재한다. 그러므로 TRAIL 내성을 극복할 수 있는 방법을 고안하는 연구는 암 치료 요법에 매우 중요하다. 본 연구에서는 SIRT1 siRNA 또는 SIRT1 inhibitor인 amurensin G를 사람 유방암세포에 처리하면 DR5및 c-Myc의 발현 증강과 c-$FLIP_{L/S}$ 및 Mcl-1 발현 억제를 유도하므로서, TRAIL 에 내성을 나타내는 사람유방암세포 MCF-7 세포의 TRAIL 감수성을 증강시킴을 알 수 있었다. 또한, SIRT1 억제에 의한 caspase 활성화, PARP cleavage 및 Bcl-2 발현감소를 나타내었다. 이러한 연구결과는 SIRT1 저해에 의한 DR5 유도와 함께 c-FLIP 발현 억제가 TRAIL 내성 암세포의 TRAIL 반응성 증강에 유용한 기전으로 사용 될 수 있음을 시사하였다.

• Transactions on Electrical and Electronic Materials
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• 제9권1호
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• pp.33-37
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• 2008

This paper describes the development of a new $5.0{\times}3.2mm$ SAW filter bank which is consist of 12 L, C matching components and 4 SAW bare chips on PCB substrate with CSP technology. We improved the manufacturing cost by removing the ceramic package through direct flip bonding of $LiTaO_3$ SAW bare chip on PCB board after mounting L, C passive element on PCB board. After that we realized the hermitic sealing by laminating the epoxy film. To confirm the confidentiality and durability of the above method, we have obtained the optimum flip bonding & film laminating condition, and figured out material property and structure to secure the durability & moisture proof of PCB board. The newly developed super mini $5.0{\times}3.2mm$ filter bank shows the superior features than those of existing products in confidence, electrical, mechanical characters.

• 대한용접접합학회:학술대회논문집
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• 대한용접접합학회 2007년 추계학술발표대회 개요집
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• pp.245-247
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• 2007

Sn-base solder bump is mainly used in micro-joining for flip chip package. The quantity of intermetallic compounds that was formed between Cu pad and solder interface importantly affects reliability. In this research, micro-bump was fabricated by two binary electroplating and the intermetallic compounds(IMCs) was estimated quantitatively. When the micro Sn-Ag solder bump was made by electroplating, SiC powder was added in the plating solution for protecting of intermetallic growth. Then, the intermetallic compounds growth was decrease with increase of amount of SiC power. However, if the mount of SiC particle exceeds 4 g/L, the effect of the growth restraint decrease rapidly.

• 한국마이크로전자및패키징학회:학술대회논문집
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• 한국마이크로전자및패키징학회 2002년도 International Symposium
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• pp.219-225
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• 2002

This paper presents a novel "bumpless flip chip package"for cost! performance driven devices. Using the conventional electroplating and etching processes, this package enables the production of fine pitch BGA up to 256 I/O with single layer routing. An array of circuitry down to $25-50{\mu}{\textrm}{m}$ line/space is fabricated to fan-in and fan-out of the bond pads without using bumps or substrate. Various types of joint methods can be applied to connect the fine trace and the bond pad directly. The resin-filled terminal provides excellent compliancy between package and the assembled board. More interestingly, the thin film routing is similar to wafer level packaging whereas the fan-out feature enables high lead count devices to be accommodated in the BGA format. Details of the design concepts and processing technology for this novel package are discussed. Trade offs to meet various cost or performance goals for selected applications are suggested. Finally, the importance of design integration early in the technology development cycle with die-level and system-level design teams is highlighted as critical to an optimal design for performance and cost.

• 한국식품영양과학회지
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• 제40권9호
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• pp.1201-1207
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• 2011

TRAIL은 최근 암세포의 apoptosis 유도를 위한 효율적인 도구로 제시되었으나 많은 암세포들이 TRAIL 저항성을 획득한 것으로 알려져 TRAIL 저항성 극복을 위한 새로운 방법론의 제시가 요구되어지고 있다. Genistein은 대두의 대표적인 생리활성 물질인 isoflavonoid의 일종으로 많은 암세포에서 G2/M arrest를 유발하면서 apoptosis를 유도하는 것으로 알려져 있다. 본 연구에서는 U937 인체백혈병세포를 대상으로 genistein에 의한 TRAIL 유도 apoptosis의 감수성 증대 여부를 조사하였다. 본 연구의 결과에 의하면 U937 세포에서 세포독성이 없는 범위의 genistein 처리는 TRAIL에 의한 apoptosis 유도를 매우 증진시켰으며, 이는 tBid의 발현 증가와 cFLIPL의 발현 감소와 연계된 caspase의 활성 증가와 연관성이 있었다. 또한 caspase의 활성 저해제는 genistein과 TRAIL의 복합처리에 의한 apoptosis를 유의적으로 감소시켜 복합 처리에 의한 apoptosis의 유도에 caspase의 활성 증대가 필수적임을 알 수 있었다. 따라서 genistein은 TRAIL 저항성을 획득한 암세포의 효율적인 combined therapy approach를 위해 유용하게 사용될 수 있음을 알 수 있었다.

• 생명과학회지
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• 제26권4호
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• pp.431-438
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• 2016

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 세포질 cytochrome 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.