Interleukin-4 receptor-targeted delivery of Bcl-xL siRNA sensitizes tumors to chemotherapy and inhibits tumor growth

Guruprasath, Padmanaban,Kim, Jihoon,Gunassekaran, Gowri Rangaswamy,Chi, Lianhua,Kim, Soyoun,Park, Rang-Woon,Kim, Sang-Hyun,Baek, Moon-Chang,Bae, Sang Mun,Kim, Sang-Yeob,Kim, Dong-Kyu,Park, In-Kyu,Kim, Elsevier 2017 Biomaterials Vol.142 No.-

<P><B>Abstract</B></P> <P>IL-4 receptor (IL-4R) is commonly up-regulated on tumor cells, and interactions between the receptor and Interleukin-4 (IL-4) can induce the expression of anti-apoptotic proteins, including Bcl-xL. This contributes to tumor cell survival and their resistance to chemotherapy. In this study, we exploited IL-4R-targeted delivery of Bcl-xL siRNA to IL-4R-expressing tumor cells in order to sensitize them to chemotherapy. To target IL-4R, an IL-4R-binding peptide, IL4RPep-1, was attached to branched polyethyleneimine-superparamagnetic iron oxide nanoparticles (BPEI-SPION). These nanoparticles were then complexed with Bcl-xL-targeting siRNA. IL-4R-targeted BPEI-SPION/Bcl-xL siRNA more efficiently reduced Bcl-xL gene expression and enhanced cytotoxicity of doxorubicin in MDA-MB231 breast tumor cells compared to untargeted BPEI-SPION/Bcl-xL siRNA. The siRNA was released from the complexes after 15 h of incubation at pH 5.5 and was stable in the complexes up to 72 h in the serum. The IL-4R-targeted BPEI-SPION/siRNA was internalized by cells through IL-4R, successfully escaped the endosomes, and was dispersed into the cytoplasm. Near-infrared fluorescence and magnetic resonance imaging demonstrated that <I>in vivo</I> tumor homing and accumulation of IL-4R-targeted BPEI-SPION/siRNA were both higher than untargeted BPEI-SPION/siRNA. The IL-4R-targeted BPEI-SPION/Bcl-xL siRNA, in combination with doxorubicin, significantly inhibited tumor growth in mice compared to untargeted BPEI-SPION/Bcl-xL siRNA. These results suggest that the IL-4R-targeted delivery of Bcl-xL siRNA to IL-4R-expressing tumors can sensitize tumors to chemotherapy and enhance the efficacy of anti-tumor therapeutics.</P> <P><B>Graphical abstract</B></P> <P>Interaction between IL-4 and IL-4R provides downstream signaling for the expression of Bcl-xL and inhibits doxorubicin-induced apoptosis. IL4RPep-1-labeled BPEI-SPION/Bcl-xL siRNA selectively binds to IL-4R-expressing tumor cells and is internalized into the cells through the receptor-mediated endocytosis, which contributes to the silencing of the Bcl-xL gene expression. This sensitizes IL-4R-expressing tumor cells to doxorubicin and enhances its therapeutic efficacy.</P> <P>[DISPLAY OMISSION]</P>

Ectopic expression of Bcl-2 or Bcl-xL suppresses p-fluorophenylalanine-induced apoptosis through blocking mitochondria-dependent caspase cascade in human Jurkat T cells

한규현,오현지,전도연,김영호,Han, Kyu-Hyun,Oh, Hyun-Ji,Jun, Do-Youn,Kim, Young-Ho Korean Society of Life Science 2003 생명과학회지 Vol.13 No.1

Phenylalanine의 구조유사체인 p-fluotophenylalanine (FPA)은 인체 급성백혈병세포주인 Jurkat T 세포의 세포자살을 유도한다. FPA에 의한 세포자살에 미치는 Bcl-2 또는 Bcl-xL의 영향을 조사하기 위해, Bcl-2 또는 Bcl-xL을 stable transfection하거나 empty vectors만을 Transfection한 Jurkat 세포를 이용하여 FPA의 세포독성과 FPA에 의한 세포내 세포자살 신호전달경로를 비교 분석하였다. Jurkt T 세포에 0.63∼3.0 mLf의 FPA를 처리하였을 때 세포의 생육도는 농도에 비례하여 감소하였다. 또한 세포자살관련 DNA fragmentation, caspase-8 activatoin, Bid cleavage, mitochondria로 부터의 cytochrome c 방출, caspase-9 및 -3 activation, PARP degradation 등이 유도되었다. 한편, FPA에 의해 유도되는 이러한 일련의 생화학적 현상들은 Bcl-2 또는 Bcl-xL의 overexpression에 의해 현저히 저해되었다. 이상의 결과들은 caspase-8 activation, Bid cleavage, mitochondnal cytochrome c 방출에 의해 활성화되는 casuase cascade 등의 현상이, Bcl-2 또는 Bcl-xL에 의해 억제됨을 나타내며 FPA에 의해 유도되는 세포자살에 필요한 과정임을 시사한다. $\rho$-Fluorophenylalanine (FPA), a phenylalanine analog, is able to induce apoptotic cell death of human acute leukemia Jurkat T cells. To better understand the mechanism by which FPA induces apoptotic cell death, the effect of ectopic expression of antiapoptotic proteins, Bcl-2 and Bcl-xL, on FPA-induced apoptosis was investigated by employing lurkat T cells transfected with Bcl-2 gene (JT/Bcl-2) or Bcl-xL gene (1/Bcl-xL) and Jurkat T cells transfected with vector (JT/Neo or J/Neo). When Jurkat T cells, JT/Neo or J/Neo, were exposed to FPA at concentrations ranging from 0.63 to 5.0 mM, the cell viability determined by MTT assay declined in a dose-dependent manner. In addition, apoptotic DNA fragmentation along with several apoptotic events such as caspase-8 activation, Bid cleavage, mitochondrial cytochrome c release, caspase-9 activation, caspase-3 activation, and degradation of PARP was induced. However, the FPA-induced cytotoxic effect, activation of caspase-8, and cleavage of Bid were significantly abrogated by ectopic expression of Bcl-2 or Bcl-xL. At the same time, there was marked reduction in the level of cytochrome c release from mitorhondria, caspase-9 activation, caspase-3 activation, and degradation of PARP. These results indicate that caspase-8 activation, Bid cleavage, and mitochondrial cytochrome c release with subsequent activation of the caspase cascade are negatively regulated by Bcl-2 or Bcl-xL, and are thus required for FPA-induced apoptosis in Jurkat T cells

GFP를 이용하여 in-vivo에서 추적한 Bad와 Bcl-XL의 Mitochondria 이동

윤수한,김진영,박승우,안영환,안영민,조기홍,조경기,Yoon, Soo Han,Kim, Jin Young,Park, Seung Woo,Ahn, Young Hwan,Ahn, Young Min,Cho, Ki Hong,Cho, Kyung Gi 대한신경외과학회 2000 Journal of Korean neurosurgical society Vol.29 No.10

Objectives : The subcellular locations of Bad, Bid, Bax and Bcl-XL change during apoptosis and this change is important for the regulation of cell death. The purpose this study was to elucidate binding of Bad with Bcl-XL in vivo Methods : We mads Bad with Green Fluorescent Protein(GFP) using PCR method. We transfected and overexpressed GFP-Bad with or without Bcl-XL cotransfection in living COS-7 cell. Results : Bad and Bcl- XL bind one another in healthy living cells and this association controled mitochondrial docking. In the absence of Bad-XL, Bad was mainly cytosolic and partially bound to mitochondria. Upon coexpression of Bad and Bcl-XL, most of Bad translocated to mitochondria. These should suggest that Bad binds to the mitochondrial and cytoplasmic forms of Bcl-XL and Bad bound to cytoplasmic Bcl-XL translocates to mitochondria. These in vivo findings confirm that Bad make a complexes with Bcl- XL and cause mitochondrial translocation of Bad-Bcl-XL complex.

일산화질소에 의해 유도된 연골 세포 사멸에서의 항세포사멸유전자 Bcl-XL의 방어 효과

한창환 ( Chang Whan Han ),신윤학 ( Yun Hak Shin ),권순용 ( Soon Yong Kwon ),조윤경 ( Yun Kyoung Cho ),지보근 ( Bo Keun Jee ),김영율 ( Young Yul Kim ),김순희 ( Soon Hee Kim ),박기숙 ( Ki Sook Park ),강길선 ( Gil Son Khang ) 한국조직공학과 재생의학회 2005 조직공학과 재생의학 Vol.2 No.2

To evaluate whether transfection of human chondrocytes with an anti-apoptotic gene, Bcl-XL, can prevent nitric oxide (NO)-induced chondrocyte apoptosis. We stably transduced early passage chondrocytes with an anti- apoptotic gene Bcl-XL and retrovirus. Bcl-XL transducants were compared with chondrocytes transduced in parallel with a Lac-Z gene. After the cells were treated with 500 microM of SNP, the process of apoptosis was assessed by trypan blue exclusion, enzyme-linked immunosorbent assay (ELISA), and flow cytometry. The functional aspect of the chondrocyte was measured by proteoglycan synthesis assay. Chondrocytes were cultivated in NO, the chondrocytes that were either not transfected or transfected with Lac-Z exhibited a decrease in viable cell number in comparison to the Bcl-XL transfected chondrocytes. The NO significantly increased fragmentation of DNA in chondrocytes that were either not transfected or transfected with Lac-Z, and this increase in DNA fragmentation was significantly greater than in the chondrocytes transfected with Bcl-XL. Flow cytometric result showed that apoptotic chondrocytes were significantly higher in the transfected and Lac-Z transfected chondrocytes than Bcl-XL transfected chondrocytes. Chondrocytes transfected with Bcl-XL were protected from NO-induced impairment of proteoglycan synthesis response to NO. From the present study, we have demonstrate that NO-induced chondrocytes death involve mechanisms subjected to regulation by an anti-apoptotic protein, Bcl-XL. Therefore, Bcl-XL gene therapy has the potential to protect apoptosis in human chondrocyte.

Role of phospholipase D2 in anti-apoptotic signaling through increased expressions of Bcl-2 and Bcl-xL

Oh, Kyoung-Jin,Lee, Sung-Chang,Choi, Hye-Jin,Oh, Doo-Yi,Kim, Sang-Chul,Min, Do Sik,Kim, Jung Mogg,Lee, Ki Sung,Han, Joong-Soo Wiley Subscription Services, Inc., A Wiley Company 2007 Journal of cellular biochemistry Vol.101 No.6

<P>We have previously reported that Fas-resistant A20 cells (FasR) have phospholipase D (PLD) activity upregulated by endogenous PLD2 overexpression. In the present study, we investigated how overexpressed PLD2 in FasR could generate survival signals by regulating the protein levels of anti-apoptotic Bcl-2 and Bcl-xL. To confirm the effect of PLD2 on Bcl-2 protein levels, we transfected PLD2 into wild-type murine B lymphoma A20 cells. The transfected cells showed markedly the increases in Bcl-2 and Bcl-xL protein levels, and became resistant to Fas-induced apoptosis, similar to FasR. Treatment of wild-type A20 cells with phosphatidic acid (PA), the metabolic end product of PLD2 derived from phosphatidylcholin, markedly increased levels of anti-apoptotic Bcl-2 and Bcl-xL proteins. Moreover, PA-induced expressions of Bcl-2 and Bcl-xL were enhanced by propranolol, an inhibitor of PA phospholydrolase (PAP), whereas completely blocked by mepacrine, an inhibitor of phospholipase A<SUB>2</SUB> (PLA<SUB>2</SUB>), suggesting that PLA<SUB>2</SUB> metabolite of PA is responsible for the increases in Bcl-2 and Bcl-xL protein levels. We further confirmed the involvement of arachidonic acid (AA) in PA-induced survival signals by showing that 1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA), PA without AA, was unable to increase Bcl-2 and Bcl-xL proteins. Moreover, PA notably increased cyclooxygenase (COX)-2 protein expression, and PA-induced expression of both Bcl-2 and Bcl-xL was inhibited by NS-398, a specific inhibitor of COX-2. Taken together, these findings demonstrate that PA generated by PLD2 plays an important role in cell survival during Fas-mediated apoptosis through the increased Bcl-2 and Bcl-xL protein levels which resulted from PLA<SUB>2</SUB> and AA-COX2 pathway. J. Cell. Biochem. 101: 1409–1422, 2007. © 2007 Wiley-Liss, Inc.</P>

Role of Bcl-xL/Beclin-1 in synergistic apoptotic effects of secretory TRAIL-armed adenovirus in combination with mitomycin C and hyperthermia on colon cancer cells

Kim, Seog-Young,Lee, Dae-Hee,Song, Xinxin,Bartlett, David L.,Kwon, Yong Tae,Lee, Yong J. Springer US 2014 Apoptosis Vol.19 No.11

<P>In this study, we attempted to develop a multimodality approach using chemotherapeutic agent mitomycin C, biologic agent tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo-2L), and mild hyperthermia to treat colon cancer. For this study, human colon cancer LS174T, LS180, HCT116 and CX-1 cells were infected with secretory TRAIL-armed adenovirus (Ad.TRAIL) and treated with chemotherapeutic agent mitomycin C and hyperthermia. The combinatorial treatment caused a synergistic induction of apoptosis which was mediated through an increase in caspase activation. The combinational treatment promoted the JNK-Bcl-xL-Bak pathway which transmitted the synergistic effect through the mitochondria-dependent apoptotic pathway. JNK signaling led to Bcl-xL phosphorylation at serine 62, dissociation of Bak from Bcl-xL, oligomerization of Bak, alteration of mitochondrial membrane potential, and subsequent cytochrome <I>c</I> release. Overexpression of dominant-negative mutant of Bcl-xL (S62A), but not dominant-positive mutant of Bcl-xL (S62D), suppressed the synergistic death effect. Interestingly, Beclin-1 was dissociated from Bcl-xL and overexpression of dominant-negative mutant of Bcl-xL (S62A), but not dominant-positive mutant of Bcl-xL (S62D), suppressed dissociation of Beclin-1 from Bcl-xL. A combinatorial treatment of mitomycin C, Ad.TRAIL and hyperthermia induced Beclin-1 cleavage, but the Beclin-1 cleavage was abolished in Beclin-1 double mutant (D133A/D146A) knock-in HCT116 cells, suppressing the apoptosis induced by the combination therapy. We believe that this study supports the application of the multimodality approach to colon cancer therapy.</P>

A novel mechanism of irinotecan targeting MDM2 and Bcl-xL

Lee, Boah,Min, Jeong A.,Nashed, Abdullateef,Lee, Sang-Ok,Yoo, Jae Cheal,Chi, Seung-Wook,Yi, Gwan-Su Elsevier 2019 Biochemical and biophysical research communication Vol.514 No.2

<P><B>Abstract</B></P> <P>Irinotecan is a strong anticancer drug whose mechanism of action has been reported only for the inhibition of DNA topoisomerase I (Topo I) through its active metabolite SN-38. In this study, we present a new mechanism of Irinotecan which inhibits the activities of MDM2, an E3 ligase of tumour suppressor p53, and Bcl-xL, an anti-apoptotic protein, through direct binding. In our structure modelling study, Irinotecan could fit to the binding sites of MDM2 and Bcl-xL for their known drugs, Nutlin-3 and ABT-737, with a better binding affinity than to Topo I. The direct binding of Irinotecan to both proteins was confirmed through a NMR study. We further showed that Irinotecan increased the amount of p53 only in the presence of MDM2 and inhibited the physical interaction of Bcl-xL with Bim, a core pro-apoptotic protein. In addition, we demonstrated that Irinotecan induced the down regulation of proliferation and strong G2/M arrest in HCT116 colon cancer cells shortly after treatment. Collectively, we suggest a new mechanism of action for Irinotecan as a dual target inhibitor of MDM2 and Bcl-xL facilitating the anticancer activities mediated by p53 and Bcl-xL interaction partners.</P> <P><B>Highlights</B></P> <P> <UL> <LI> The direct binding of Irinotecan to MDM2 and Bcl-xL was confirmed in modelling and NMR. </LI> <LI> The effects of Irinotecan in MDM2 were confirmed by cell cycle, proliferation, and p53 expression. </LI> <LI> The effects of Irinotecan in Bcl-xL were confirmed by apoptosis and Bcl-xL-Bim binding. </LI> <LI> Irinotecan regulates proliferation, apoptosis and induces strong G2/M arrest. </LI> </UL> </P>

기저세포암 및 편평세포암에서 Cyclin D1과 Bcl-xL의 발현

권준일 ( Jun Il Kwon ),김상표 ( Sang Pyo Kim ),이규석 ( Kyu Suk Lee ),조재위 ( Jae We Cho ) 대한피부과학회 2011 대한피부과학회지 Vol.49 No.3

Background: Cyclin D1 has a key function in cell proliferation. Bcl-xL has anti-apoptotic effects that can protect against cell death. However, it is still largely unknown whether these proteins play important roles in development of basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). Objective: The purpose of this study was to investigate the expression of cyclin D1 and Bcl-xL in both of these carcinomas. Methods: In this study, we examined expression levels of cyclin D1 and Bcl-xL in both carcinomas by immunohistochemistry, immunofluorescence and Western blot analysis. Results: We found increased expression of both proteins in BCC and SCC using immunohistochemistry. Moreover, expression of cyclin D1 and Bcl-xL were increased in both carcinomas relative to normal skin or seborrheic keratosis in Western blot analysis. BCC showed greater increases in expression of cyclin D1 and Bcl-xL on peripheral lesions compared to the center of tumor nests. Conclusion: That expression of cyclin D1 and Bcl-xL were increased in BCC and SCC, suggests that progression of the cell cycle combined with inhibition of apoptosis contributes to the carcinogenesis of BCC and SCC. (Korean J Dermatol 2011;49(3):234∼241)

자연과학편 : 트레드밀 운동 중 경사도 조절에 의한 운동강도가 쥐의 세포사멸 관련 유전자에 미치는 영향

김재호(JaeHoKim),김용안(YongAnKim) 한국체육학회 2007 한국체육학회지 Vol.46 No.6

본 연구는 경사도 증가에 의한 운동강도에 따라 실험쥐의 골격근에 아몹토시스 관련 유전자인 caspase-3, bcl-2, bax, bcl-xl, bad, DNA fragmentation 변화를 알아보는데 목적이 있다 0%, 10%, 20% 경사도로 나누어 각각 50, 40, 30분 간 운동(속도고정; 2Qm/min)을 수행시켜 다음과 같은 결론을 얻었다. caspase-3, Bcl-2, Bad, Bax, bcl-xl mRNA, DNA fragmentation 은 다른 그룹과 비교하여 20% 경사도에서 가장 높았다. 20% 경사도 그룹내 변화에서 운동직후 에 가장 높게 증가한 것은 caspase-3, Bcl-2, bcl-xl이고, 회복기 5h에 가장 높게 증가한 것은 Bad, Bax, DNA fragmentation이다. 따라서 근수축 부하의 증가에 따라 아품토시스 관련 유전자의 변화는 차이가 있으며, 부하가 증 가할수록 아폽토시스에 의한 세포사멸이 나타난다. Exercise is associated with intensity-dependent muscle strengts and microinjury disturbances. Apoptosis may contribute to this phenomenon investigated the effects of different intensities determined by graded treadmill exercise on skeletal muscle of mice. The caspase-3, Bcl-2, Bax, Bcl-XL, Bad transcript contents were estimated by RT-PCR DNA laddering was used to detect DNA fragmentation as an indicator of apoptosis. The results of the study can summerized as follows ; caspase-3, Bcl-2, Bad, Bax, bcl-xl mRNA and DNA fragmentation was significantly peaked at 20% degree exercise group when compared with 0% and 10% degree exercise group. Also, the caspase-3, Bcl-2, bcl-xl at 20% degree exercise group was peaked that immediately when compared with rest, recovery 5h. Bad, Bax, DNA fragmentation at 20% degree exercise group was peaked that recovery 5h when compared with rest, immediately. It was concluded that load of muscle contraction increased that undergo cell death by apoptosis.

Fluoxetine Up-Regulates Bcl-xL Expression in Rat C6 Glioma Cells

최미란,오동훈,김석현,양병환,Jun-Seok Lee,최준호,Hyun-Soo Jeon,채영규,박용천 대한신경정신의학회 2011 PSYCHIATRY INVESTIGATION Vol.8 No.2

Objective To analyze both differentially expressed genes and the Bcl-xL protein expression after acute and chronic treatment with fluoxetine in rat C6 glioma cells. Methods C6 glioma cells were cultured for 24 h or 72 h after treatment with 10 μM fluoxetine, and gene expression patterns were observed using microarray and qRT-PCR. Then, cells were cultured for 6 h, 24 h, 72 h or 96 h after treatment with 10 μM fluoxetine, and the expression of Bcl-xL protein was measured using western blot. Results As determined by microarray, treatment with fluoxetine for 24 h up-regulated 33 genes (including Bcl-xL and NCAM140)and down-regulated 7 genes (including cyclin G-associated kinase). Treatment with fluoxetine for 72 h up-regulated 53 genes (including Gsαand Bcl-xL) and down-regulated 77 genes (including Gαi2 and annexin V). Based on the qRT-PCR results, there was an increase in GsαmRNA and a decrease in Gαi2 mRNA at 72 h in fluoxetine-treated cells as compared to control, a result that was consistent with microarray. We also observed an increase in Bcl-xL mRNA (both at 24 h and at 72 h) in fluoxetine-treated cells as compared to control, demonstrating a tendency to increase gradually. Bcl-xL protein expression increased as the duration of fluoxetine treatment increased. Conclusion These results suggest that chronic treatment with fluoxetine not only initiates the cAMP pathway through inducing Gsα expression but also induces Bcl-xL expression, thus inhibiting apoptosis.