Síntese, avaliação biológica e modelagem molecular de potenciais antitumorais análogos da sanguinarina planejados por simplificação molecular / Synthesis, biological evaluation and molecular modeling of potential antitumors analogues from sanguinarine designed by molecular simplification

A sanguinarina é um alcaloide capaz de inibir Bcl-xL, uma proteína antiapoptótica que se encontra superexpressa em linhagens tumorais e que está frequentemente relacionada à resistência destas frente a quimioterápicos antineoplásicos. No intuito de identificar potenciais agentes antitumorais, o objetivo deste trabalho foi sintetizar três séries de análogos da sanguinarina planejados por simplificação molecular e avaliar sua atividade biológica. Dez N-benzil-naftil-aminas (3a-e; 4a-e) e dez arilisoquinolinas (6a-e; 7a-e) foram sintetizadas em duas a três etapas reacionais, utilizando-se métodos de aminação redutiva e acoplamento de Suzuki. Insucesso na etapa de reação de Heck impossibilitou a síntese da terceira série, benzofenantridínica, apesar de testadas diversas condições reacionais. Avaliação da citotoxicidade em linhagens de glioblastoma U87MG revelou que a série N-benzilnaftil-amina apresenta melhor atividade quando comparada às aril-isoquinolinas, sendo para ambas, observada atividade superior à temozolamida, principal fármaco para o tratamento de glioblastoma. Estudos em linhagem não tumorigênica MRC-5 demonstraram que os análogos foram significativamente superiores à sanguinarina em relação à seletividade. Os compostos mais mais promissores, 4a e 6e, induziram morte celular por apoptose e causaram despolarização da membrana mitocondrial, indicando morte apoptótica pela via extrínseca. Ademais, 4a interrompeu o ciclo interrompeu o ciclo celular na fase G2/M, indicando que o mesmo seria um agente ciclo celular específico. Simulações de dinâmica molecular sugerem que os compostos interagem com a proteína Bcl-xL principalmente por interações hidrofóbicas, e que o composto 4a apresentaria afinidade com o alvo semelhante à sanguinarina, embora esta tenha apresentado atividade superior em células U87. Perspectivas incluem estudos das vias de indução de morte celular, além da expansão do painel de células. Conclui-se, portanto, que os análogos da sanguinarina representam um arcabouço a ser explorado pelos químicos medicinais no desenvolvimento de potenciais antineoplásico

Sanguinarine is an alkaloid able to inhibit Bcl-xL, an antiapoptotic protein which is overexpressed in tumor cells and related to their resistance against antineoplastic chemotherapy. Regarding to develop potential antitumor agents, the aim of this work was the synthesis of three series of sanguinarine analogues designed by molecular simplification and their biological evaluation. Ten N-benzyl-naphtyl-amines (3a-e; 4ae) and ten aryl-isoquinolines (6a-e; 7a-e) were synthesized in two or three reaction steps through reductive amination and Suzuki coupling. Failure about Heck-type reaction had impaired the synthesis of the thirth series, benzophenanthridine, although several conditions were tested. Cytotoxicity evaluation against U87MG glioblastoma cell line showed that N-benzyl-naphtyl-amines are more active than aryl-isoquinolines and both series were superior to temozolamide, the main drug for glioblastoma treatment. Tests against non-tumorigenic cell MRC-5 indicated that the analogues were significantly superior to sanguinarine regarding selectivity. The most promising compounds, 4a e 6e, induced cell death by apoptosis and mitochondrial membrane depolarization, indicating apoptotic death by extrinsic pathway. 4a provide cell cycle arrest at G2/M phase, suggesting that it is a specific cell cycle agent. Molecular dynamics suggested that compounds interact with Bcl-xL mainly by hydrophobic interactions and 4a has affinity to the protein like sanguinarine, although the last showed superior activity against U87 cells. Perspectives include mechanistics studies about cell death pathway and expanding cell panel. In conclusion, sanguinarine anlogues represent a scaffold to be explored by medicinal chemists to the development of potential antitumor agent

miR-133a mediates Bcl-xl expression to regulate endothelial cell apoptosis / 医学研究生学报

ObjectiveThe specific mechanism of microRNA-133a (miR-133a) involved in the pathological process of atherosclerosis (As) remains an open question. This study aims to explore the role of miR-133a in the regulation of endothelial cell apoptosis.MethodsCultured human coronary endothelial cells (HCAECs) were treated with oxidized low density lipoprotein (ox-LDL). The cell viability was detected by MTT assay. The mRNA levels of Bcl-xl and miRNA (miR-133a, etc) were detected by qRT-PCR method. The expression of anti-apoptotic protein Bcl-xl and cleaved-caspase3 was detected by Western blotting, and the apoptosis rate was detected by flow cytometry. The transient transfection technique was used to observe the effect of overexpression and silencing of miR-133a, on the expression of target gene Bcl-xl protein and endothelial cell apoptosis.Results Ox-LDL was observed to decrease the viability of HCAECs cells and induce HCAECs apoptosis; miR-133a increased abnormally in the apoptosis model; after silencing miR-133a, the decrease of Bcl-xl and the increase of apoptosis rate induced by ox-LDL were partially reversed; the overexpression of miR-133a, Bcl-xl decreased and the apoptosis rate increased, and the difference was statistically significant.Conclusion miR-133a might target and regulate the anti-apoptotic protein Bcl-xl, to induce endothelial cell apoptosis and promote the formation of AS.

Eye-acupuncture intervention reduces cerebro-cortical apoptosis of neurovascular unit in cerebral ischemia-reperfusion injury rats / 针刺研究

OBJECTIVE: To observe the effect of eye-acupuncture intervention on cerebro-cortical apoptosis of microvascular endothelial cells, neurons and astrocytes (main components of neurovascular unit) and the expression of Bad (an apoptosis promoter) and B-cell lymphoma-extra large(Bcl-xL) proteins in focal cerebral ischemia-reperfusion injury (CIRI) rats, so as to explore its mechanisms underlying improvement of CIRI. METHODS: SD rats were randomly divided into control, sham-operation, model 3 h, model 24 h, model 72 h, eye-acupuncture 3 h, eye-acupuncture 24 h and eye-acupuncture 72 h groups(n＝12 in each group). The CIRI model was established by middle cerebral artery occlusion/reperfusion (MCAO/R). Eye-acupuncture was applied to bilateral "Gan" (Liver) regions, "Shen" (Kidney) regions, "Shangjiao" (Upper-energizer) and "Xiajiao" (Lower-energizer) for 20 min, once 3 h and every 12 h after modeling. The expression levels of Bad and Bcl-xL in the ischemic cerebral cortex tissue were detected by Western blot. The apoptotic neurons, microvascular endothelial cells and astrocytes were assayed by immunofluorescence double labeling (Nestin/TUNEL, CD34/TUNEL and glial fibrillary acidic protein [GFAP]/TUNEL) separately. RESULTS: After modeling, the numbers of apoptotic neurons, microvascular endothelial cells and astrocytes in the ischemic cerebral cortex tissue were significantly increased in the model 72 h group than in the sham-operation group (P<0.01). Following the treatment, the numbers of the 3 types of apoptotic cells were markedly lower in the eye-acupuncture 72 h group than in the model 72 h group(P<0.01). The expression levels of Bad and Bcl-xL proteins were notably up-regulated in the model 3 h, model 24 h and model 72 h groups than in the sham operation group(P<0.01). Following eye-acupuncture intervention, modeling induced increase of the Bad expression were obviously reversed in eye-acupuncture 24 h and eye-acupuncture 72 h groups than those in the 2 model groups(P<0.05). And the increase of Bcl-xL expression levels were further increased in the eye-acupuncture groups in comparison with those in the 3 model groups (P<0.01). CONCLUSION: Eye-acupuncture can down-regulate the expression of Bad protein, and up-regulate the expression of Bcl-xL protein in the ischemic cerebral cortex in CIRI rats, which may contribute to its function in reducing apoptotic neurons, microvascular endothelial cells and astrocytes, suggesting a protective effect of eye-acupuncture intervention on neurovascular unit.

Long Noncoding RNA HEIH Promotes Colorectal Cancer Tumorigenesis via Counteracting miR-939-Mediated Transcriptional Repression of Bcl-xL / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Studies have found that long noncoding RNA HEIH (lncRNA-HEIH) is upregulated and facilitates hepatocellular carcinoma tumor growth. However, its clinical significances, roles, and action mechanism in colorectal cancer (CRC) remains unidentified. MATERIALS AND METHODS: lncRNA-HEIH expression in CRC tissues and cell lines was measured by quantitative real-time polymerase chain reaction. Cell CountingKit-8, ethynyl deoxyuridine incorporation assay, terminal deoxynucleotidyl transferase dUTP nick end labeling staining, and nude mice xenografts assays were performed to investigate the roles of lncRNA-HEIH. RNA pull-down, RNA immunoprecipitation, chromatin immunoprecipitation, and luciferase reporter assays were performed to investigate the action mechanisms of lncRNA-HEIH. RESULTS: In this study, we found that lncRNA-HEIH is significantly increased in CRC tissues and cell lines. lncRNA-HEIH expression is positively associated with tumor size, invasion depth, and poor prognosis of CRC patients. Enhanced expression of lncRNA-HEIH promotes CRC cell proliferation and decreases apoptosis in vitro, and promotes CRC tumor growth in vivo. Whereas knockdown of lncRNA-HEIH inhibits CRC cell proliferation and induces apoptosis in vitro, and suppresses CRC tumor growth in vivo. Mechanistically, lncRNA-HEIH physically binds to miR-939. The interaction between lncRNA-HEIH and miR-939 damages the binding between miR-939 and nuclear factor κB (NF-κB), increases the binding of NF-κB to Bcl-xL promoter, and promotes the transcription and expression of Bcl-xL. Moreover, Bcl-xL expression is positively associatedwith lncRNA-HEIH in CRC tissues. Blocking the interaction between lncRNA-HEIH and miR-939 abolishes the effects of lncRNA-HEIH on CRC tumorigenesis. CONCLUSION: This study demonstrated that lncRNA-HEIH promotes CRC tumorigenesis through counteracting miR-939-mediated transcriptional repression of Bcl-xL, and suggested that lncRNA-HEIH may serve as a prognostic biomarker and therapeutic target for CRC.

Quercetin enhances 5-fluorouracil-induced apoptosis in gastric cancer by down-regulating c-Jun expression / 中国病理生理杂志

AIM:To investigate the enhancing effect of quercetin on the 5-fluorouracil-induced apoptosis in gastric cancer.METHODS: MTT assay was conducted to evaluate the effect of quercetin on the 5-fluorouracil-induced death of gastric cancer cell line BGC-823.Co-immunoprecipitation and Western blot analysis were performed to detect the expression of c-Jun and Bcl-xL,phosphorylation of c-Jun,activation of caspase-9 and caspase-3,and release of cytochrome C in BGC-823 cells treated with quercetin and 5-fluorouracil.The apoptosis of BGC-823 cells treated with quercetin and 5-fluorouracil was analyzed by flow cytometry.RESULTS: Adjuvant therapy of quercetin significantly enhanced the 5-fluorouracil-induced death of BGC-823 cells.Meanwhile, quercetin decreased the half maximal inhibitory concentration (IC50)of 5-fluorouracil to BGC-823 cells.Quercetin treatment significantly inhibited the expression of c-Jun,and inhibited the 5-fluorouracil-induced phosphorylation of c-Jun and the interaction between c-Jun and activating transcription factor 2 (ATF2).Subsequently,quercetin inhibited the up-regulation of Bcl-xL induced by 5-fluorouracil in the BGC-823 cells. However,transfection with c-Jun plasmid abolished the promoting effect of quercetin on 5-fluorouracil-induced cell death. In addition, quercetin promoted 5-fluorouracil-induced release of cytochrome C from mitochondria and caspase-dependent apoptosis in BGC-823 cells.CONCLUSION:Quercetin treatment enhances 5-fluorouracil-induced mitochondrial apoptosis in BGC-823 cells through c-Jun/ATF2/Bcl-xL pathway.

Cancer-associated-fibroblasts regulate the chemoresistance of lung cancer cell line A549 via SDF-1 secretion / 中华肿瘤杂志

Objective@#To investigate whether cancer-associated- fibroblasts (CAF), the key component of tumor microenvironment, regulate the chemoresistant capacity of lung cancer cell line A549 through SDF-1 secretion.@*Methods@#Primary cell isolation techniques was used to isolate cancer-associated-fibroblasts from lung cancer patients. MTT assay was applied to determine the proliferation and chemoresistance of A549 cells. Quantative PCR was used to detect the mRNA changes of Bcl-xL. Western blotting was used to detect the protein expression of Bcl-xL. ELISA was applied to detect the SDF-1 secretion from normal fibroblasts (NF) and CAF.@*Results@#CAF promoted the proliferation of A549 cells, while NF had no significant effect on them. After 72 hrs incubation, the absorbance value of A549+ CAF medium group was 0.814±0.006, significantly different from the 0.753±0.006 of the A549+ NF medium group (P<0.05). The Q-PCR assay indicated that mRNA expressions of Bcl-xL in the A549 group, A549+ NF medium group and A549+ CAF medium group were 1.00±0.11, 1.10±0.09 and 3.50±0.30, respectively, showing a significant difference between the A549+ NF medium group and A549+ CAF medium group (P<0.05). The Western blot showed that protein expressions of Bcl-xL in the A549 group, A549+ NF medium group and A549+ CAF medium group were 1.00±0.08, 1.10±0.12 and 3.10±0.25, respectively, with a significant difference between the A549+ NF medium group and A549+ CAF medium group (P<0.05). The ELISA results showed that the SDF-1 concentrations in the A549+ NF medium group and A549+ CAF medium group were 3.23±0.02 and 9.53±0.10, respectively, significantly different from each other (P<0.05). The MTT assay indicated that the absorbance values of OD of A549 group, A549+ AMD3100 group, A549+ NF medium group, A549+ NF medium+ AMD3100 group, A549+ CAF medium and A549+ CA Fmedium+ AMD3100 group were 0.43±0.03, 0.25±0.02, 0.48±0.03, 0.31±0.03, 0.72±0.06 and 0.45±0.03, respectively. The data of A549+ NF medium group was significantly different from that of A549+ CAF medium group (P<0.05).@*Conclusions@#Cancer-associated-fibroblasts enhance the drug resistance of A549 cells through SDF-1 secretion, upregulating the expression level of Bcl-xL through interaction with CXCR4. Our study not only illustrates that tumor microenvironment is able to enhance drug resistance of tumor, but also provides experimental evidence for the cancer-associated-fibroblasts as a potential therapeutic target for the treatment of lung cancer.

STAT3 Activation Way Blocking Effects on Colorectal Cancer cells HT29 Growth / 中国现代普通外科进展

Objective:Explore decoy oligonucleotide (ODN) technology targeted blocking STAT3 activation way effects on colorectal cancer HT29 cell growth,to provide experimemt basis for gene therapy of colorectal cancer.Methods:Using decoy ODN in vitro transfect colorectal cancer HT29 cell line,to targeted blocking STAT3 activation way.Fluorescence microscope and flow cytometry detect transfection conditions and efficiency,and MTT method detect the cell growth inhibition,Realtime RT-PCR and Western blotting test STAT3 in cells,the Bcl-xl and Caspase3 mRNA and protein expression.Results:STAT3 decoy ODN can efficient transfection into colorectal cancer cells,and is due to the nucleus.HT29 cell in the STAT3 decoy ODN,growth inhibition rate increased significantly,and mRNA and quantity of protein expression of STAT3 significantly reduce (P＜0.05),while caspase3 increase significantly (P＜0.05).Conclusion:Named Decoy ODN targeted after blocking STAT3 activation way,which can effectively cut colorectal cancer cells HT29 STAT3 gene expression,and inhibit the growth of the cells.The mechanism may be related to lower the expression of STAT3 and the Bcl-xl and Caspase3.

Promotion of combination of chrysin and camptothecin on apoptosis of CNE2 cells / 中草药

objective To research whether the combination of chrysin and camptothecin can promote the apoptosis of human nasopharyngeal carcinoma cell line CNE2 and to explore the molecular mechanism of the combinative effect. Methods: CNE2 cells were pretreated with designed dose of chrysin (10|, 20, and 40 μmol/L) for 2 h, then treated with camptothecin (1 μg/mL) for 24 h. The morphologic changes were observed under inversed microscope and the cell viability was measured using MTT. The activity of caspase-3 and PARP, which was regarded as the protein marks of apoptosis, was determined by Western blotting. Then the cells were treated with chrysin for different time and the time course of apoptosis inhibitory protein, Bcl-xL was also detected using Western blotting. Results: Increases of cell death were observed in the group with combined chrysin and camptothecin, but no obvious cell death could be found in chrysin, camptothecin alone, and control groups; The data of cell viability supported this results; With the enhance of pretreatment dose of chrysin, the cell viability decreased. There were the significant differences between the combined groups and the control one (P<0.05), and between the combined groups and both the chrysin and camptothecin groups separately (P<0.05). Chromatin condensation, which was the indication of apoptosis, could be observed when the cells were stained with Hochest 33342; The proprotein of caspase-3 and PARP degraded and there were the dose-dependent and time-dependent effect. The pan-caspase inhibitor Z-VAD-fmk could inhibit the apoptosis of CNE2 cells which were treated with the combination of chrysin and camptothecin, according to the cell viability and the activation of caspase-3 and PARP; The time-dependent down-regulation in the apoptosis inhibitory protein Bcl-xL could be observed. Conclusion: The cotreatment of chrysin and camptothecin could promote the apoptosis of CNE2 and the down-regulation of apoptosis inhibitory protein Bcl-xL played an important role in the combinative effect.

Effect of Electrical Stimulation to Cerebellar Fastigial Nucleus on Cerebral Ischemia-reperfusion Injury in Rats / 中国康复理论与实践

@#Objective To investigate the effect of electrical stimulation to cerebellar fastigial nucleus on expression of nuclear factor-kappa B (NF-кB) P50, tumor necrosis factor-α (TNF-α) and Bcl-xL mRNA in rats brain after cerebral ischemia-reperfusion. Methods Sprague-Dawley rats were randomly divided into normal control group (NC group), cerebral ischemia-reperfusion group (I/R group), fastigial nucleus stimulation (FNS) group, and fastigial nucleus lesion (FNL) group. A focal cerebral ischemia-reperfusion model was established with middle cerebral artery occlusion (MCAO). 7 and 14 days after operation, the infarct volume was measured, and the protein of NF-кB P50 in rats brain was detected with Western blotting; the expression of TNF-α and Bcl-xL mRNA was detected with RT-PCR. Results Compared with I/R group, the expression of NF-кB P50 protein increased in FNS group (P<0.05), with the decrease of expression of TNF-α mRNA (P<0.01) and increase of Bcl-xL mRNA (P<0.05), while the infarct size decreased (P<0.01). There was no significant difference between FNL group and I/R group for all the measurements (P>0.05). Conclusion FNS could induce the expression of P50 protein and Bcl-xL mRNA, and inhibit the expression of TNF-α mRNA, and reduce infarct size, which may associated with the neuroprotection of central nervous system from injury.

Effect of Electrical Stimulation to Cerebellar Fastigial Nucleus on Cerebral Ischemia-reperfusion Injury in Rats / 中国康复理论与实践

Objective To investigate the effect of electrical stimulation to cerebellar fastigial nucleus on expression of nuclear fac-tor-kappa B (NF-кB) P50, tumor necrosis factor-α(TNF-α) and Bcl-xL mRNA in rats brain after cerebral ischemia-reperfusion. Methods Sprague-Dawley rats were randomly divided into normal control group (NC group), cerebral ischemia-reperfusion group (I/R group), fasti-gial nucleus stimulation (FNS) group, and fastigial nucleus lesion (FNL) group. A focal cerebral ischemia-reperfusion model was established with middle cerebral artery occlusion (MCAO). 7 and 14 days after operation, the infarct volume was measured, and the protein of NF-кB P50 in rats brain was detected with Western blotting;the expression of TNF-αand Bcl-xL mRNA was detected with RT-PCR. Results Com-pared with I/R group, the expression of NF-кB P50 protein increased in FNS group (P0.05). Conclusion FNS could induce the expression of P50 protein and Bcl-xL mRNA, and inhibit the expression of TNF-αmRNA, and reduce infarct size, which may associated with the neuroprotection of central ner-vous system from injury.

Knockdown of Bcl-xL Enhances Growth-Inhibiting and Apoptosis-Inducing Effects of Resveratrol and Clofarabine in Malignant Mesothelioma H-2452 Cells

Mcl-1 and Bcl-xL, key anti-apoptotic proteins of the Bcl-2 family, have attracted attention as important molecules in the cell survival and drug resistance. In this study, we investigated whether inhibition of Bcl-xL influences cell growth and apoptosis against simultaneous treatment of resveratrol and clofarabine in the human malignant mesothelioma H-2452 cells. Resveratrol and clofarabine decreased Mcl-1 protein levels but had little effect on Bcl-xL levels. In the presence of two compounds, any detectable change in the Mcl-1 mRNA levels was not observed in RT-PCR analysis, whereas pretreatment with the proteasome inhibitor MG132 led to its accumulation to levels far above basal levels. The knockdown of Bcl-xL inhibited cell proliferation with cell accumulation at G2/M phase and the appearance of sub-G0/G1 peak in DNA flow cytometric assay. The suppression of cell growth was accompanied by an increase in the caspase-3/7 activity with the resultant cleavages of procaspase-3 and its substrate poly (ADP-ribose) polymerase, and increased percentage of apoptotic propensities in annexin V binding assay. Collectively, our data represent that the efficacy of resveratrol and clofarabine for apoptosis induction was substantially enhanced by Bcl-xL-lowering strategy in which the simultaneous targeting of Mcl-1 and Bcl-xL could be a more effective strategy for treating malignant mesothelioma.

Activation of protease-activated receptor 2 inhibits apoptosis of lung cancer cells / 中国药理学通报

Aim To investigate the activation of prote-ase-activated receptor 2 ( PAR2 ) in regulation of the expression of epidermal growth factor receptor ( EGFR) and apoptosis of lung cancer ( LC) cells. Methods LC cells A549 and its EGFR-silenced counterpart were incubated in the medium added with tryptase. Quanti-tative RT-PCR and Western blotting were used to de-tect the expression of EGFR in A 5 4 9 cells . The apop-tosis and Bax/Bcl-xL of cells were also recorded. Re-sults Treating A549 cells with tryptase in the culture for 48 hrs resulted in a marked increase in the expres-sion of EGFR in A549 cells. Marked decreases in a tryptase dose-dependent manner in apoptotic A549 cells were detected in the presence of tryptase. Expo-sure to tryptase markedly decreased the levels of Bax and increased the levels of Bcl-xL in A549 cells, which were not shown in EGFR-deficient A549 cells. Conclusion Tryptase can increase the expression of EGFR in LC cell line, A549 cells, which protects A549 cells from apoptosis, increases Bcl-xL, and sup-presses Bax in A549 cells.

Effect of Recombination Human Erythropoietin on the Apoptosis of Glutamate-induced Retinal Cells / 华中科技大学学报(医学版)

Objective To study the protective effect of recombination human erythropoietin (rhEPO) on the apoptosis of retinal neurons induced by glutamate.Methods The primary retinal neurons of postnatal SD rats were cultured in vitro for 7 days and divided into 3 groups :control group ,glutamate group and rhEPO pretreatment group.The neurons in the rhEPO pre-treatment group were afterwards allocated to three subgroups in terms of different rhEPO treatments (0.15 ,0.30 or 0.50 U/mL rhEPO for 12 h).Those in glutamate group and rhEPO pretreatment group were treated with glutamate at the concentration of 20μmol/L for 30 min for establishment of the apoptosis model.Twenty-four h later ,the apoptosis index (AI) was assayed by TUNEL and the expressions of BCL-xL mRNA and protein was detected by RT-PCR and immunocytochemistry respective-ly.Results The AI was significantly higher in the glutamate group than in the control group (P<0.01).The AI was signifi-cantly reduced ,and the expression level of BCL-xL mRNA and protein was markedly dose-dependently increased in the rhEPO pretreatment groups compared with the glutamate group (P<0.01).Conclusion The rhEPO pretreatment can inhibit the glu-tamate-induced apoptosis of retinal neurons by up-regulating the expression of BCL-xL .

The effect of siRNA targeted against Bcl-xl on the expression of apoptosis relevant factors Bcl-xl,Bax, Caspase-3 / 中华风湿病学杂志

Objective To investigate the effect of siRNA targeted against Bcl-xl on cell proliferation of rheumatoid arthritis (RA) and the effect on expres-ion of apoptosis relevant factors Bcl-xl,Bax,Caspase-3.Methods Human RA synovial cells were cultured and passed by tissue block collagenase digestion method.The siRNA plasmid targeting Bcl-xl gene was constructed by Bcl-xl cDNA sequence provided by gene banks,while single missense sequences were used as negative controls.LipofectamineTM 2000 was used to transfect synovial cells.The effect of Bcl-xl silencing on proliferation of synovial cells was evaluated by MTT at 24,48,72 hours after transfection.The expressions of Bcl-xl,Bax,Caspase-3 protein,synovial apoptosisrelated factors were determined by Western blotting after transfected at different time points.The average of multiple-sample average was analyzed by single-factor x2 test or LSD-t and Tamhane's T2 test was used for twotwo comparison.Results MTT result s showed that RNA interference that specifically silent Bcl-xl could obviously suppress the proliferation of sliding film cells,which was most eveident at 48 hours.This inhibition was gradually weakened with prolonged time,but when compared was the control group,differences was significant (P＜0.05).Western blotting results displayed that when compared to the control group,Bcl-xl protein expression obviously declined after transfection P＜0.01,which was the least at 48 h time point.Bax,Caspase3 protein expression were obviously increased when compared to the coutrol group.Conclusion The expression of Bcl-xl,a RA synovial cell anti-apoptotic factor,is significantly reduced by specific RNA interference silencing Bcl-xl,which may play an important role in inhibiting the excessive proliferation of synovial cells.

Aberrant expression of TCTP protein inhibits cell apoptosis by up-regulating Bcl-xL in human & nbsp;pancreatic cancer / 中国癌症杂志

10.3969/j.issn.1007-3969.2013.05.001

Using Bcl-xL anti-apoptotic protein for altering target cell apoptosis

Background: Altering target cell apoptosis is one of the challenging ideas of biotechnological applications. There are several applications of over expressing Bcl-xL anti-apoptotic protein from recombinant protein production to DNA vaccination strategies. The aim of the present study is to evaluate the anti-apoptotic efficacy of Bcl-xL expressing dual promoter plasmid system as a candidate to be used for recombinant protein production and DNA vaccination approaches. For this purpose, Bcl-xL anti-apoptotic protein gene was inserted in a dual expressing vector system in frame with EGFP (enhanced green fluorescence protein) after IRES (internal ribosomal site). The plasmid has a multiple cloning site after CMV (cytomegalovirus promoter) left empty to be inserted a biopharmaceutical protein gene region or DNA vaccine antigens. Results: In order to determine the anti-apoptotic efficacy of Bcl-xL inserted dual expressing vector, BHK-21 cells were transfected both with this plasmid and empty vector as control. Apoptosis was stimulated by several apoptosis inducing agents and serum deprivation in the transfected cells for 48 hrs. Cells expressing Bcl-xL protein in frame with EGFP were determined by flow cytometry as an indicator of cell viability. Additionally, apoptosis were determined by intracellular cleaved Casp 3 staining in Bcl-xL expressing EGFP positive cells. The dual expression plasmid bearing Bcl-xL anti-apoptotic protein prolonged the cell survival rate and protected cells from apoptosis upon apoptosis induction by doxorubicin and camptothecin in which the anti-apoptotic efficacies are inhibited through over expressing of Bcl-xL. pIRES2EGFP/Bcl-xL transfected cell ratio was significantly higher compared to empty vector transfected cells (P < 0.001). In contrast, apoptotic cell ratio was significantly lower in pIRES2EGFP/Bcl-xL transfected cell population compared to empty vector transfected cells (P < 0.001). Conclusion: In conclusion...

Effect of rhDCN on proliferation of leukemic cell line K562 / 白血病·淋巴瘤

ObjectiveTo investigate the anti-tumorigenesis function of rhDCN on the leukemia K562 cells in vitro and analyze the possible mechanism.Methods Exponential phase of K562 cells were transfected with pcDNA3.1(+)-DCN,and PBS,liposome alone,and pcDNA3.1(+) vector were as control groups.Morphology change of K562 cells was detected by Wright stain,and cell proliferation activity was detected by MTT. Cell cycle and apoptosis of K562 cells were assessed by FCM. The expression of apoptosis-related protein,including bcl-xl,Mcl-1 and Bax were detected by Western blot.ResultsWright stain showed that typical apoptotic morphology of K562 cells were observed in DCN transfected group.There were no morphological changes of apoptosis in other groups. MTT method results showed that proliferation inhibition rate of the transfected cells [24 h (16.14±1.08) ％,48 h (14.07±1.01) ％,72 h (20.29±1.19) ％]was higher than that of the other control groups (P ＜ 0.05).FCM results showed that the apoptosis index (20.15±1.31) ％of the DCN transfected group was higher than that of the other groups (P ＜ 0.05),and most of cells arrested in the G0/G1 phase (51.15±0.57) ％ (P ＜ 0.05).Western blot results showed the expression levels of Bax were increased while bcl-xl and Mcl-1 were decreased in pcDNA3.1(+)-DCN/K562 group.ConclusionrhDCN can inhibit the growth of K562 cells and induce the apoptosis.The effect of DCN on bcl-xl,Mcl-1,Bax may play a role in its mechanism.

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

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 microM 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 microM 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 Gsalpha and Bcl-xL) and down-regulated 77 genes (including Galphai2 and annexin V). Based on the qRT-PCR results, there was an increase in Gsalpha mRNA and a decrease in Galphai2 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 Gsalpha expression but also induces Bcl-xL expression, thus inhibiting apoptosis.

Expression of Cyclin D1 and Bcl-xL in Basal Cell Carcinoma and Squamous Cell Carcinoma / 대한피부과학회지

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.

Effects of sleep deprivation on depressive behaviors and hippocampus bcl-xl in rats treated with chronic stresses / 中华行为医学与脑科学杂志

Objective To explore the changes of Bcl-xl levels and activities in hippocampus resulting from sleep deprivation, and then to reveal the mechanism for rapid antidepressant aroused by sleep deprivation.Methods 40 SD rats were randomly divided into normal control group, chrinic stress group, sleep deprivation group and tank contral group.10 rats in each group.The depression animal model was established by chronic mild unpredictable stress(CUMS) methods.Sleep deprivation was preformed by the modified multiple platform method ( MMPM ).The animal model and the effect of antidepressant were evaluated by the open field test.The expression levels of Bcl-xl were separately observed by immunohistochemical technology in hippocampus CA1 ,CA3 and DG.Results 1.Compared with the normal control rats, ambulation ( 35.30 ± 18.77,81.30 ± 18.41, P ＜ 0.01 ) and rearing (20.50 ±4.84,27.70 ± 8.19, P＜0.05 ) increased ,and the stopping time in the center decreased (4.60 ± 1.35,2.20 ± 1.55, P ＜ 0.01 ) in the CUMS depressant animal model.2.The Bcl-xl average values of optical density (OD) in hippocampus CA1 ,CA3 ,DG of the model group was lower than that of the normal control group significantly (0.1356 ±0.0224,0.1389 ±0.0250,0.1457 ±0.0162;0.1725 ±0.0327,0.1734 ±0.0261,0.1768 ±0.0271; P＜0.01 ) ,and that of the sleep deprivation group was higher than that of the model group (0.1621 ± 0.0128,0.1603 ± 0.0137,0.1625 ± 0.0192 ;0.1356 ± 0.0224,0.1389 ± 0.0250,0.1457 ± 0.0162; P ＜ 0.05 ).Conclusion Rats showed depressive behaviors after 21 days stresses,while 72 hours sleep deprivation could reverse this effect.The up regulation of the expression and phosphorylation of Bcl-xl by sleep deprivation may participate in the antidepressant-like effect of sleep deprivation.