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Objective: To investigate the effect of Bak gene overexpression on proliferation of breast cancer MCF-7 cells and sensitivity to paclitaxel. Methods: Western blotting and Real-time PCR were used to detect the expression of Bak protein in breast cancer MCF-7 cells before and after transfection. The effects of Bak gene overexpression and paclitaxel treatment for 24 hours, 48 hours and 72 hours on MCF-7 cell proliferation and cell cycle were detected by cell counting kit-8(CCK-8) and flow cytometry. Results: The results of Western blotting and Real-time PCR showed that the expression of Bak protein increased significantly after transfection. The results of Western blotting and Real-time PCR showed that the expression of Bak protein increased significantly after transfection, and the mRNA expression of MCF-7-Bak group was 2.15±0.07, which was significantly higher than that of MCF-7-NC group, which was 1.03±0.04(r= 13.412, P <0.05). After 48 hours, 72 hours and 96 hours of transfection, the proliferation rates of MCF-7-Bak cells were (0.31 ± 0.03)%, (0.37±0.03)%, (0.47±0.04)%, respectively, which were lower than that of MCF-7 NC group (0.40± 0.03)%, (0.48±0.04)%, (0.61±0.06)%, and the difference was statistically significant (t48 = 2.145, t72 = 3.164, t96 = 5.487, P<0.05). The number of G2 phase cells in MCF-7 Bak group were (26.84±2.69)% significantly higher than that in MCF-7 NC group (16.02±1.61)% ({=12.887, P<0.05). After 24 hours, 48 hours, and 72 hours of paclitaxel treatment, the cell inhibition rate of MCF-7-Bak group was (35.98±4.00)%, (54.66±5.50) %, (80.11 ± 8.00) %, respectively, which were higher than that of MCF-7 NC group (24.12±2.40)%, (40.12±4.00)%, (61.09±6.09)%. And the difference was statistically significant (t24 = 8.456, t48 = 10.547, t72 = 13.442, P<0.05). After 24 hours of paclitaxel treatment, the number of G2 phase cells in MCF-7 Bak group was (73.01 ±7.02)% higher than that in MCF-7 NC group (63.84±6.68) %(t = 9.736, P<0.05). Conclusion: Up-regulate of Bak gene expression can inhibit the proliferation of breast cancer MCF-7 cells, up-regulating the proportion of G0/G1 phase, and enhance the sensitivity of paclitaxel.
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Objective To investigate the neuroprotective mechanism of sevoflurane in rats resuscitated from cardiac arrest (CA).Methods A ventricular fibrillation-induced CA model was established.Forty Wistar rats were randomly divided into the sham group,sevoflurane group and control group.Apoptosis-related proteins were measured by Western blot at 24 h after restoration of spontaneous circulation (ROSC).The status of mitochondrial permeability transition pore (MPTP) were measured using a spectrophotometer,and the mitochondrial membrane potential (MMP) were measured with JC-1 fluorescent probe.At 72 h after ROSC,the apoptotic index of neurons in hippocampal CA1 region was counted by TUNEL staining.Results The protein expression of Bax,Bak,cleaved-caspase 9,cleavedcaspase 3 and cytosolic cytochrome c were lower in the sevoflurane group (all P＜0.05),the protein expression of Bcl-2 was higher in the sevoflurane group compared with the control group (P＜0.05).The sevoflurane group had a less opening status of MPTP and a higher MMP compared with the control group (all P＜0.05).The sevoflurane group had less apoptotic neurons compared with the control group (P＜0.05).Conclusion By up-regulating the expression of Bcl-2,down-regulating Bax and Bak,sevoflurane could reduce the apoptosis of neurons and decrease the opening of MPTP,eventually reduce cerebral injuries.
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This study examined the effect of saponins from Tupistra chinensis Bak (STCB) on the growth of sarcoma S-180 cells in vitro and in mouse xenografts as well as the underlying mechanisms.Cell proliferation was assessed by MTT assay.Cell cycle distribution was determined by flow cytometry.Sarcoma S-180 tumor-bearing mice were treated with different doses of STCB with 10 μg/mL 5-fluorouracil (5-Fu) as a positive control.The activity of nuclear factor (NF)-κB was detected by gel mobility shift assay.The mRNA level of NF-κB was determined by real-time quantitative RT-PCR.The results showed that in vitro STCB inhibited the growth of S-18 0 cells in a concentration-dependent manner,which was accompanied by cell cycle arrest at S-phase.In vivo STCB significantly inhibited the growth of S-180 tumor mouse xenografts in a dose-dependent manner with apparent induction of cell apoptosis.Moreover,STCB inhibited the activity of NF-κB p65 and reduced the expression of NF-κB p65 mRNA in mouse xenografts.It was concluded that STCB inhibits the proliferation and cell cycle progression of S-180 cells by suppressing NF-κB signaling in mouse xenografts.Our findings suggest STCB is a promising agent for the treatment of sarcoma.
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This study examined the effect of saponins from Tupistra chinensis Bak (STCB) on the growth of sarcoma S-180 cells in vitro and in mouse xenografts as well as the underlying mechanisms.Cell proliferation was assessed by MTT assay.Cell cycle distribution was determined by flow cytometry.Sarcoma S-180 tumor-bearing mice were treated with different doses of STCB with 10 μg/mL 5-fluorouracil (5-Fu) as a positive control.The activity of nuclear factor (NF)-κB was detected by gel mobility shift assay.The mRNA level of NF-κB was determined by real-time quantitative RT-PCR.The results showed that in vitro STCB inhibited the growth of S-18 0 cells in a concentration-dependent manner,which was accompanied by cell cycle arrest at S-phase.In vivo STCB significantly inhibited the growth of S-180 tumor mouse xenografts in a dose-dependent manner with apparent induction of cell apoptosis.Moreover,STCB inhibited the activity of NF-κB p65 and reduced the expression of NF-κB p65 mRNA in mouse xenografts.It was concluded that STCB inhibits the proliferation and cell cycle progression of S-180 cells by suppressing NF-κB signaling in mouse xenografts.Our findings suggest STCB is a promising agent for the treatment of sarcoma.
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Apoptosis, especially the intrinsic mitochondrial cell death pathway, is regulated by the BCL-2 family of proteins. Defects in apoptotic machinery are one of the main mechanisms that cells employ to evade cell death and become cancerous. Targeting the apoptotic defects, either by direct inhibition of BCL-2 family proteins or through modulation of regulatory pathways, can restore cell sensitivity to cell death. This review will focus on the aspects of BCL-2 family proteins, their interactions with kinase pathways, and how novel targeted agents can help overcome the apoptotic blockades. Furthermore, functional assays, such as BH3 profiling, may help in predicting responses to chemotherapies and aid in the selection of combination therapies by determining the mitochondrial threshold for initiating cell death.
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Objective To explore the effects of Bcl-2 and BaK gene silencing on cell apoptosis ,osteogen-esis activity and free Ca2+concentration of MG-63 cell lines. Methods The siRNA sequences targeted Bcl-2 and BaK respectively were designed;Bcl-2 and BaK silencing adenovirus vector scramble RNA vector and empty vec-tor were constructed to transfect MG-63 cell lines. MTT method was used to examine cell viability;ALP and flow cytometry were conducted to observe osteogenesis activity and free Ca2+concentration. Results Bcl-2 gene silenc-ing decreased cell viability,reduced osteogenesis activity and increased free Ca2+ concentration when compared with controls but BaK gene silencing had the opposite effects. The effect of Bcl-2+BaK gene silencing on cell was similar to the empty control. Conclusions Cell apoptosis,osteogenesis activity and free Ca2+concentration of MG-63 change following Bcl-2 and BaK gene silencing,implicating their roles in osteoporosis.
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Objective To explore the effects of Bcl-2 and BaK gene silencing on cell apoptosis ,osteogen-esis activity and free Ca2+concentration of MG-63 cell lines. Methods The siRNA sequences targeted Bcl-2 and BaK respectively were designed;Bcl-2 and BaK silencing adenovirus vector scramble RNA vector and empty vec-tor were constructed to transfect MG-63 cell lines. MTT method was used to examine cell viability;ALP and flow cytometry were conducted to observe osteogenesis activity and free Ca2+concentration. Results Bcl-2 gene silenc-ing decreased cell viability,reduced osteogenesis activity and increased free Ca2+ concentration when compared with controls but BaK gene silencing had the opposite effects. The effect of Bcl-2+BaK gene silencing on cell was similar to the empty control. Conclusions Cell apoptosis,osteogenesis activity and free Ca2+concentration of MG-63 change following Bcl-2 and BaK gene silencing,implicating their roles in osteoporosis.
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Objective:To study the expression levels of nuclear factor kappa B, Bcl-2 associated K and TNF-αproteins to discuss the effects of NF-κB and Bak proteins in the pathogenesis of UC.Methods:Eighty clean grade of adult Sprague-Dawley( SD) rats were used,male and female in half and then rando mly selected sixty as the model group,another twenty as the control group.SD rats model were manufactured by a compound method:Trinitrobenzene sulfonic acid ( TNBS )+ethanol.We observed and assessed colonic mucosa by the general morphology and histological changes.To applicated immunohistochemistry and RT-PCR methods to detected the protein and mRNA expression levels of NF-κB,Bak and TNF-αin the model groups and the control group and to analysed their relationships.Results:The successful rate of making model was 97%.The number of inflammatory cells in the model groups more than the control(P0.05).The expression levels of NF-κB,TNF-αincreased as the histological grade increased(P<0.05),however,the expression level of Bak decreased(P<0.05).NF-κB in colonic mucosa of rats with UC had a significantly positive correlation with that of TNF-α(r=0.892,P<0.01),and negatively correlated with that of Bak(r=-0.793,P<0.01).Conclusion:The levels of NF-κB and Bak may be related to the occurrence and development of UC.
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Objective: To study the chemical constituents in the whole plant of Pteris deltodon. Methods: Nine compounds were isolated from 95% ethanol extract in the whole plant of P. deltodon and purified by silica gel chromatography, Sephadex LH-20, and pre-HPLC, and their structures were identified on the basis of spectroscopic data and literatures. Results: Nine compounds were isolated and elucidated as β-sitosteol (1), (22E)-5α,8α-epidioxyergosta-6,22-dien-3β-ol (2), ent-kaur-16-ene-2β,15α-diol (3), cycloart-23-en-3β,25-diol (4), cycloart-25-en-3β,24-diol (5), emodin (6), 1,7-dihydroxyxanthone (7), ursolic acid (8), and ergosterol (9). Conclusion: All the compounds are for the first time isolated from P. deltodon; Compound 4 and 5 are cycloartanes first isolated from genus Pteris L.
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Objective To explore the association of expression of apoptosis-associated gene BAK and cFLIP with the biological behaviors in endometriosis. Methods The expression of BAK and cFLIP protein gene in eutopic and ectopic tissue samples from 40 cases with pathologically confirmed ovarian endometriosis and 40 cases with pathologically confirmed normal endometrium was detected by immunohistochemical method. Results ①The expression of BAK and cFLIP protein gene was found in three groups of different endometrial tissue. ②The expression of BAK protein gene was increased gradually in ectopic endometrial , eutopic endometrium and normal tissue and there was significantly difference between every two groups ,while cFLIP was contrary expressed (P 0.05). ④The expression of BAK protein gene in severe group (Ⅲ-Ⅳ period) is lower than mild group both in eutopic or ectopic endometrial tissues,while cFLIP was contrary expressed (P < 0.05). ⑤The expression of BAK and cFLIP was negatively correlated with each other in ectopic endometrium (r=-0.389,P< 0.05). Conclusion BAK and cFLIP was negatively expressed in EMS, which may take a part in the endometrial apoptosis and disorderly proliferation. BAK and cFLIP may play an important role in the the diagnosis and treatment of endometriosis.
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Objective: To study the chemical constituents from Lepidogrammitis drymoglossoides. Methods: The constituents were isolated and purified by various column chromatographies, and their structures were identified on the basis of chemical evidences and spectroscopic analysis including MS, 1H-NMR, and 13C-NMR. Results: Fourteen compounds were isolated and identified as β-ecdysterone (1), physcion (2), emodin (3), umbelliferone (4), scoparone (5), aesculetin (6), caffeic acid (7), chlorogenic acid (8), protocatechuic acid (9), pyrocatechualdehyde (10), gallic acid (11), 4-hydroxybenzoic acid methyl ester (12), docosanyl tetracosanoate (13), and hexadecanoic acid (14). Conclusion: Compounds 3-5, 8, and 11-13 are isolated from the plants of genus Lepidogrammitis Ching for the first time.
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Paracoccidioidomycosis presents a variety of clinical manifestations and Paracoccidioides brasiliensis can reach many tissues, most importantly the lungs. The ability of the pathogen to interact with host surface structures is essential to its virulence. The interaction between P. brasiliensis and epithelial cells has been studied, with particular emphasis on the induction of apoptosis. To investigate the expression of different apoptosis-inducing pathways in human A549 cells, we infected these cells with P. brasiliensis Pb18SP (subcultured) and 18R (recently isolated from cell culture and showing a high adhesion pattern) samples in vitro. The expressions of Bcl-2, Bak and caspase 3 were analysed by flow cytometry and DNA fragmentation using the TUNEL technique. Apoptosis of human A549 cells was induced by P. brasiliensis in a sample and time-dependent manner. Using an in vitro model, our data demonstrates that caspase 3, Bak, Bcl-2 and DNA fragmentation mediate P. brasiliensis-induced apoptosis in A549 cells. The overall mechanism is a complex process, which may involve several signal transduction pathways. These findings could partially explain the efficient behaviour of this fungus in promoting tissue infection and/or blood dissemination.
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Humans , Apoptosis/physiology , Epithelial Cells/microbiology , Host-Pathogen Interactions , Lung/cytology , Paracoccidioides/physiology , /analysis , Cell Line/microbiology , Flow Cytometry , Paracoccidioides/pathogenicity , /analysis , /analysisABSTRACT
PURPOSE: This study examined the effects of Tacrolimus (FK506) on the expression of the apoptotic signal transduction proteins of Jurkat human T-lymphocytes. METHODS: The cell viability was examined by a MTT assay, DAPI stain, enzyme activity of caspase family proteins, and western blotting for Bcl-2, Bak, Fas, and Fas-L. The cells were cultured in the presence or absence of FK506. FK506 induced cell death was confirmed to be apoptosis by the observation of nuclear fragmentation. RESULTS: The viability of Jurkat cells was decreased by the addition of FK506 in a dose- and time- dependent manner. The FK506 induced activation of caspase-3 protease was observed. FK506 didn't increase the catalytic activity of caspase -6, -8, and -9 proteases of Jurkat cells in a time-dependent manner. The viability was improved when a caspase-3 inhibitor was added. However, the caspase-9 inhibitor did not affect the viability. Bak protein expression was increased, and the Bcl-2 protein was decreased for some time. The expression of Fas and Fas-L were unaffected by FK506. CONCLUSION: FK506 induces dose- and time-dependent apoptotic cell death, and enhances the apoptosis of Jurkat cell by increasing the expression of Bak and caspase-3.
Subject(s)
Humans , Apoptosis , bcl-2 Homologous Antagonist-Killer Protein , Blotting, Western , Caspase 3 , Caspase 9 , Cell Death , Cell Survival , Jurkat Cells , Peptide Hydrolases , Signal Transduction , T-Lymphocytes , TacrolimusABSTRACT
Objective To study the function of Bak in mitochondrial signaling pathway and interaction between Bax and Bak during apoptosis.Methods Bax/Bak double knock out(Bax-/- and Bak-/-)MEF cells from mouse embryo fibroblasts(MEF) and Hela cells were divided into four groups according the cell different genotypes(wild type,Bax-/-,Bak-/- and double knock out) and treated with different chemical reagents after co-transfection with Bax,Bak,Mito-Red and empty pEGFP vector.Apoptosis,mitochondrial morphology and cytochrome C release were detected with confocal microscope,immunofluoresence and Western blotting techniques.Results There were correlations between the percentage of Hela cell apoptosis and mitochondrial fission(%) as well as cytochrome C(%)(P
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BACKGROUND: Human telomerase is a ribonucleoprotein polymerase, which synthesizes telomeric repeat sequences, and human telomerase reverse transcriptase (hTERT) has been identified as the catalytic subunit, as well as the rate-limiting component, of telomerase. In this study, we attempted to identify the modulators of telomerase, and to determine the molecular mechanisms underlying cisplatin-induced apoptosis. METHODS: To determine the role of telomerase in cisplatin-induced apoptosis, we measured telomerase activity and analyzed apoptosis using PI and trypan blue staining. Also, we inhibited the caspase activations using Z-VAD-fmk to analyze the effects on expression of hTERT protein. Finally, we induced the transient co-expression of the Bcl-2 and Bak genes in HEK293 cells, and then, the telomerase activity and expression of hTERT were evaluated. RESULTS: In the Bcl-2-overexpressing HeLa cells, telomerase activity was more enhanced, and cell death was reduced to 40-50% that of the mock controls. This finding suggests that Bcl-2-induced telomerase activity exerts an antiapoptotic effect in cisplatin-induced death. As caspase activation was inhibited via Z-VAD-fmk, the hTERT protein was recovered in the mock controls, but not in the Bcl-2-overexpressing cells. This suggests that the expression of hTERT can be regulated by caspases, but Bcl-2 was located within the upstream pathway. Moreover, when the Bcl-2 and Bak genes were co-transfected into the HEK293, both telomerase activity and hTERT protein were prominently reduced. CONCLUSIONS: Bcl-2-induced telomerase activity inhibits cisplatin-induced apoptosis in HeLa cells, and can be regulated via both caspases and the interaction of Bcl-2 and Bak.
Subject(s)
Humans , Apoptosis , Caspases , Catalytic Domain , Cell Death , Cisplatin , HEK293 Cells , HeLa Cells , Ribonucleoproteins , Telomerase , Trypan BlueABSTRACT
Objective To study the roles of apoptosis genes bcl 2, bax, and bak in the pathogenesis of hepatitis D. Methods Expressions of HDAg, bcl 2, bax, and bak in liver specimens of 77 patients with hepatitis D were studied by immunohistochemical method. Meanwhile, the relationship of HDAg expression with the expressions of bcl 2, bax, and bak was studied by double labelling. Results Bcl 2 was mainly expressed in the cytoplasm of hepatocytes, and bax and bak mainly in the cytoplasm of hepatocytes and partly in the nucleus of hepatocytes, and HDAg mainly in the nucleus of hepatocytes. Lots of HDAg and bax/bak positive cells were distributed in infiltrating lymphocytes at the periportal region especially at the advancing edges of areas of piecemeal necrosis. Apoptosis of many hepatocytes was found to locate near the HDAg positive cells. There was positive correlation between the expression of bax/bak and HDAg expression ( P
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Objective To investigate the influence of iodine on apoptosis of thyrocytes and to study the effect of iodine on the pathogenesis of thyroid diseases. Methods Normal human thyrocytes were cultured in the absence or presence of 10 -8 ～10 -4 mol/L NaI. Apoptosis, Fas expression, Bcl-2 and Bak expression and Fas and soluble Fas (sFas) mRNA levels in thyrocytes were detected by flowcytometry, Western blot and semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) respectively, sFas was detected in supernatant of cultured thyrocytes by ELISA. Results (1) Low concentration of iodine (10 -8 mol/L) could inhibit apoptosis, while high concentrations of iodine (10 -6 ～10 -4 mol/L) increased apoptosis (P
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Objective To observe the effects of bak gene on killing MDR bladder cancer cells and to study its mechanisms. Methods Bak gene was transfected into MDR bladder cancer cells by liposome.The mRNA of bak and bcl 2 were detected by in situ hybridization.The protein of bak and bcl 2 were detected by SABC immunohistochemistry. The growth rate of human bladder cancer cells was studied by constructing the growth curve,cell apoptosis being observed by flow cytometry,and the outline of cells observed by fluorescence stain. Results The expression of bak mRNA was positive in EJ/bak cells (64%, P
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OBJECTIVE: In the management of degenerative conditions of the lumbar spine, spinal fusion is a popular management option and posterior interbody fusion is gaining wide acceptance for the treatment of segmental instability, spondylolisthesis, and discogenic pain. Many methods have been described, including use of autograft or allograft bone, in either structural or nonstructural form, with or without additional fixation. METHOD: The authors retrospectively analyzed 102 cases of posterior lumbar interbody fusion with BAK cage from March 1993 to April 1998. All patients have been followed for 24 to 56 months. Postoperative clinical and radiological changes are evaluated by Mcnab criteria and dynamic lumbar spine lateral measurement. RESULTS: Stable bony fusion was accomplished in 81.9% of patients at 12 months, in 87.4% of patients at 24months, and in 91.2% of patients at 3 years after surgery and their overall outcome was also remarkable(excellent: 42.2%, good: 49.1%). Postoperative correction of slipping was average 3.1mm in spondylolisthesis group. Seventy-eight percent of the previously employed patients returned to work by 24 months after surgery, and 94% were working at 3 years after surgery. CONCLUSION: The results of this study strongly imply that the BAK cage is safe and effective in the management of certain forms of degenerative conditions of lumbar spine. However proper patient selection is critical and experienced and properly trained spinal surgeons should perform this procedure.