Study on the Anti-apoptotic Effect of Curcumin on H 2O2-induced H 9c2 Cardiomyocyte Injury and the Regulation of NF-κB Signaling Pathway / 中国药房

OBJECTIVE：To investigate the anti-apoptotic effect of curcumin on H 2O2-induced H 9c2 cardiomyocyte injury and the regulation of NF-κB signaling pathway. METHODS：H9c2 cardiomyocyte were randomly divided into normal control group ， injury model group ，curcumin low-dose ，medium-dose and high-dose groups （25，50，100 μmol/L）. Normal control group didn ’t received any intervention. The cells in injury model group were induced with 50 μmol/L H2O2 for 12 h to establish the injury model. The cells in curcumin groups were treated with relevant concentration of drugs for 24 h，and then induced with 50 μmol/L H2O2 for 12 h. After cultured for 24 h，survival rate and apoptotic rate of cells were measured by MTT method and TUNEL method ；SOD activity and MDA content were determined by WTS- 8 assay and color test ；relative fluorescence intensity of LC 3 positive expression was detected by immunofluorescence method ；mRNA expression of NF-κB p65 in cells was detected by real-time PCR ； Western blotting assay was used to detect the protein expression of NF-κB p65 and p-NF-κB p65 in cells. RESULTS ：Compared with normal control group ，survival rate and SOD activity were decreased significantly in injury model group ，while apoptotic rate ， MDA content ，relative fluorescence intensity of LC 3 positive expression ，mRNA expression of NF-κB，protein expression of NF-κ B p 65 and p-NF-κB p65 as well as p-NF-κB/NF-κB were increased significantly（P＜0.05）. Compared with injury model group ， survival rates and SOD activities were increased significantly in curcumin groups ，while apoptotic rates ，MDA contents ，relative fluorescence intensities of LC 3 positive expression ，mRNA expression of LC 3 positive cells ，protein expression of NF-κB p65 and p-NF-κ B p65 as well as p-NF-κ B p65/NF-κ B p65 were decreased significantly （P＜0.05）. CONCLUSIONS ：Curcumin can increase the survival rate of H 2O2-induced H 9c2 cardiomyocyte injury ，decrease its apoptotic rate ，increase SOD activity and decrease MDA content in cardiomyocytes. Above effects may be related to the regulation of NF-κB signaling pathway.

Menthol enhances interleukin-13-induced synthesis and secretion of mucin 5AC in human bronchial epithelial cells / 南方医科大学学报

OBJECTIVE@#To investigate the effect of interleukin (IL) -13 combined with cold stimulation on synthesis and secretion of mucin (MUC) 5AC in human bronchial epithelial cell line 16HBE and explore the role of transient receptor potential 8 (TRPM8) and anti-apoptotic factor B-cell lymphoblast-2 (Bcl-2) in this process.@*METHODS@#16HBE cells were stimulated with 10 ng/mL IL-13, 1 mmol/L menthol, or both (1 mmol/L menthol was added after 6 days of IL-13 stimulation), and the changes in the expression of MUC5AC, intracellular Ca@*RESULTS@#The mRNA and protein expressions of MUC5AC increased significantly in 16HBE cells following stimulation with IL-13, menthol, and both (@*CONCLUSIONS@#Menthol combined with IL-13 produces a synergistic effect to promote the synthesis and secretion of MUC5AC in 16HBE cells possibly by activating TRPM8 receptor to upregulate the expression of Bcl-2.

Advances in pharmacological effects of higenamine on cardiovascular system / 药学学报

Higenamine (HG) is an active cardiotonic component isolated from Aconite. Chinese and foreign scholars have done a lot of research on the metabolism and pharmacological effects of HG, which confirmed that it has cardiovascular pharmacological effects of cardiactonic action and vasodilation for the treatment of heart failure and bradycardia, anti-oxidative and anti-apoptotic effects which can be used to protect the heart and reduce heart ischemia and reperfusion injury. In addition, HG inhibits the expression of iNOS mRNA by inhibiting the activity of the transcription factor NF-κB, inhibits the lipopolysaccharide (LPS)-induced NO product, and inhibits platelet aggregation and thrombus formation, thereby improving the experimental septic shock in animals. This article reviews the recent progress in cardiovasular pharmacology of HG, which will contribute to the further development and clinical application of it in the future.

Protective effects of methane-rich saline on mice with allergic asthma by inhibiting inflammatory response, oxidative stress and apoptosis / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Background: Asthma is a common cause of breathing difficulty in children and adults, and is characterized by chronic airway inflammation that is poorly controlled by available treatments. This results in severe disability and applies a huge burden to the public health system. Methane has been demonstrated to function as a therapeutic agent in many diseases. The aim of the present study was to explore the effect of methane-rich saline (MRS) on the pathophysiology of a mouse model of asthma and its underlying mechanism. Methods: A murine model of ovalbumin (OVA)-induced allergic asthma was applied in this study. Mice were divided into three groups: a control group, an OVA group, and OVA-induced asthmatic mice treated with MRS as the third group. Lung resistance index (RI) and dynamic compliance (Cdyn) were measured to determine airway hyper-responsiveness (AHR). Haematoxylin and eosin (H&E) staining was performed and scored to show histopathological changes. Cell counts of bronchoalveolar lavage fluid (BALF) were recorded. Cytokines interleukin (IL)-4, IL-5, IL-13, tumor necrosis factor α (TNF-α), and C-X-C motif chemokine ligand 15 (CXCL15) from BALF and serum were measured by enzyme-linked immunosorbent assay (ELISA). The oxidative stress indexes, including malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), myeloperoxidase (MPO), and 8-hydroxydeoxyguanosine (8-OHdG), were determined using commercial kits. Apoptosis was evaluated by western blot, quantitative real-time polymerase chain reaction (qRT-PCR), and biochemical examination. Results: MRS administration reversed the OVA-induced AHR, attenuated the pathological inflammatory infiltration, and decreased the cytokines IL-4, IL-5, IL-13, TNF-α, and CXCL15 in serum and BALF. Moreover, following MRS administration, the oxidative stress was alleviated as indicated by decreased MDA, MPO, and 8-OHdG, and elevated SOD and GSH. In addition, MRS exhibited an anti-apoptotic effect in this model, protecting epithelial cells from damage. Conclusions: Methane improves pulmonary function and decreases infiltrative inflammatory cells in the allergic asthmatic mouse model. This may be associated with its anti-inflammatory, antioxidative, and anti-apoptotic properties.

Expression of BAG3 protein in colon cancer tissue detected by tissue microarray method and its clinical significance / 吉林大学学报(医学版)

Objective:To explore the expression of Bcl related anti-apoptotic protein 3 (BAG3 protein)in colon cancer and its clinical significance,and to clarify the relationship between its expression and the occurrence and development of colon cancer.Methods:A total of 90 cases of colon cancer tissue and paracancerous tissue were selected, the expressions of BAG3 protein in different tissues were detected by tissue microarray and immunohistochemical SP method;Kaplan-Meier method,univariate analysis and multivariate analysis were used to investigate the relationship between the clinicopathological parameters and the prognosis of the patients with colon cancer.Results:The expression of BAG3 protein in the colon cancer tissue was high,and the positive expression rates of BAG3 protein in the colon cancer tissue and paracancerous tissue were 37.8% and 0.01%,respectively, and the difference was significant (P < 0.05).The expression of BAG3 protein was associated with gender and tumor size (P <0.05),but there was no correlation between the expression of BAG3 protein in the colon cancer tissue and age,pathologic grade,TNM stage,and lymph node metastasis (P >0.05).The Kaplan-Meier results showed that the expression of BAG3 protein in the colon cancer tissue was not related with the prognosis of the patients and the survival rates of the younger patients and the higher TNM staging patients were high. Conclusion:The expression of BAG3 protein in the colon cancer tissue is high,and its expression is associated with the tumor size;BAG3 protein may act as a tumor marker and therapeutic target for colon cancer.

Protective effect of BAG-1L mediated by lentivirus in human neuroblastoma cells induced by hypoxia / 中华危重病急救医学

Objective To investigate the protective effects of lentivirus mediated Bcl-2-associated athanogene 1L (BAG-1L) over-expression on human neuroblastoma cells (SH-SYSY) induced by hypoxia/re-oxygenation,and to study its effect on the phosphoinositide 3 kinase serine/threonine protein kinase (PI3K/AKT) pathway.Methods SH-SYSY cells were cultured in vitro,and the cells at logarithmic phase were collected,and they were divided into recombined lentiviral infection group [infected by lentivirus containing BAG-1L and green fluorescent protein (GFP) gene],vector control group (infected by lentivirus containing GFP without BAG-1L gene) and cell control group (non-infection).Western Blot was used to detect the expression of BAG-1L in target cells after infection for 48 hours.SH-SY5Y cells were subjected to hypoxia for 8 hours and re-oxygenation for 24 hours,then the cell counting kit-8(CCK-8) was used to detect the cell activity,and the apoptosis was detected by flow cytometry after allophycocyanin labeled annexin V/7-amino actinomycin D (Annexin V-APC/7-AAD) staining.Western Blot was used to detect the protein expressions of BAG-1L,heat shock protein 70 (HSP70),AKT and phosphorylated AKT (p-AKT).Results After infection for 48 hours,exogenous BAG-1L protein bands were observed in recombined lentiviral infection group,but not observed in cell control group and vector control group.After hypoxia/re-oxygenation treatment,the cell viability in recombined lentiviral infection group was significantly higher than that in cell control group and vector control group (A value:0.689 ± 0.036 vs.0.425 ± 0.013,0.400 ± 0.012),apoptosis was significantly decreased [apoptosis rate:(26.97 ± 1.82)％ vs.(36.60± 1.45)％,(35.77 ± 3.74)％],the protein levels of BAG-1L,HSP70 and p-AKT were significantly increased [BAG-1L protein (gray value):2.405 ± 0.167 vs.0.529 ± 0.141,0.601 ± 0.099;HSP70protein (gray value):0.997±0.123 vs.0.634±0.091,0.584±0.106;p-AKT protein (gray value):1.234±0.118 vs.0.661 ± 0.210,0.712 ± 0.199,all P ＜ 0.01],but the protein level of AKT was slightly increased (gray value:1.103 ± 0.269vs.0.646 ± 0.188,0.791 ± 0.326) without statistically significant differences (both P ＞ 0.05).There was no significant difference in all parameters between cell control group and vector control group (all P ＞ 0.05).Conclusion Lentivirus mediated BAG-1L gene over-expression can protect nerve cells against hypoxic injury and apoptosis,and the protective effect may be related to the activation increase of pathway on PI3K/AKT.

Effect of microRNAs 224 and 21 on human glioblastoma stem cell survival and the possible molecular mechanisms / 中国生化药物杂志

Objective To explore the effect of microRNAs 224 and 21 on human glioma stem cells survival and the possible molecular mechanisms.Methods qPCR was used to detect the dysregulated expression of microRNAs in malignant glioma samples, human GBM stem cells, artificially established GBM stem cell lines and human tissues.Caspase 3/7 assay, Annexin V apoptosis/fluorescence assay were performed to determine the effect of miR-21 or miR-224 mimics and inhibitor on cell apoptosis.Living cells count was used to assess miR-21 or miR-224 mimics and inhibitor on cell growth.TargetScan was used to explore potential targets of miR-21 and miR-224, and dual luciferase reporter assay was used to identify whether the 3’UTR of Caspase 3, Caspase 9 and Bim mRNA was a binding target of miR-21 or miR-224.Western blot was used to detect the expression of Caspase 3, Caspase 9 and Bim protein after transfection of miR-21 or miR-224 mimics or inhibitors.Results miR-21 and miR-224 are strongly upregulated in GSC samples, multiple GBM human tumor specimens, and GBM neurosphere stem cell lines ( P<0.05 ) .Caspase 3/7 assay and Annexin V apoptosis/fluorescence assay results showed that miR-224 and miR-21 regulated GSC apoptosis.Living cells count results demonstrated that miR-224 and miR-21 regulated GSC growth.miR-224 and miR-21 regulate pro-apoptotic gene expression by directly targeting Caspase 3, Caspase 9, and Bim 3’-UTRs. Conclusion These results indicate that miR-224 and miR-21 are important physiologic drivers of GSC resistance to apoptosis, providing new points of therapeutic leverage against these treatment-resistant cells.

Crucial Residues Modulating Interface of hBcl-B - hBaxBH3 Heterodimer as Probed by Computational Methods

ABSTRACT Cancerous cells develop resistance to cell death by over expression of anti-apoptotic proteins, which are specific to interact with pro-apoptotic and BH3-only proteins of Bcl-2 family. Delineating crucial residues mediating the heterodimer complexes (anti-apoptotic proteins - pro-apoptotic/BH3-only proteins) is indispensable to develop specific antagonists to anti-apoptotic proteins. In these backgrounds, we have herein reported crucial residues of hBaxBH3 and hBcl-B (an anti-apoptotic protein specifically interacts with human Bax but does not interact with human Bak) for hetero dimerization of the polypeptides and as well validated the structural determinants of the polypeptides through variety of virtual 'alanine mutants' and 'switch mutants' by using an array of computational methods. Residues such as D53, S60, E61, K64, E69 and D71 of hBaxBH3 and R45, H50, F53, F54, Y57, M71, S74, V75, R86, V88, T89, F93 and F159 of hBcl-B were found to be crucial residues of the polypeptides for intermolecular interaction leading hetero dimerization. Moreover, 'pharmacophoric residues' for the hBaxBH3 and hBcl-B have also been figured out and rationalized.

Apoptotic Effect of Macrophages against Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) causing tuberculosis as an intracellular pathogen initially infects alveolar macrophages following aerosol inhalation. Thus, macrophages play a critical role in the establishment of Mtb infection and macrophage cell death, a common outcome during Mtb infection, may initiate host- or pathogen-favored immune responses, resulting in facilitating protection or pathogenesis, respectively. In addition, virulent Mtb strains are known to inhibit apoptosis and consequently down-regulates immune response using a variety of strategies. In many recent studies have shown that virulent Mtb can either augment or reduce apoptosis by regulating expression of pro-apoptotic and anti-apoptotic proteins belonging to Bcl-2 family proteins. In this review, we will discuss and dissect the apoptotic pathways of Bcl-2 family proteins in Mtb-infected macrophages.

Mesenchymal stem cells: environmentally responsive therapeutics for regenerative medicine

Mesenchymal stem cells (MSCs) are partially defined by their ability to differentiate into tissues including bone, cartilage and adipose in vitro, but it is their trophic, paracrine and immunomodulatory functions that may have the greatest therapeutic impact in vivo. Unlike pharmaceutical treatments that deliver a single agent at a specific dose, MSCs are site regulated and secrete bioactive factors and signals at variable concentrations in response to local microenvironmental cues. Significant progress has been made in understanding the biochemical and metabolic mechanisms and feedback associated with MSC response. The anti-inflammatory and immunomodulatory capacity of MSC may be paramount in the restoration of localized or systemic conditions for normal healing and tissue regeneration. Allogeneic MSC treatments, categorized as a drug by regulatory agencies, have been widely pursued, but new studies demonstrate the efficacy of autologous MSC therapies, even for individuals affected by a disease state. Safety and regulatory concerns surrounding allogeneic cell preparations make autologous and minimally manipulated cell therapies an attractive option for many regenerative, anti-inflammatory and autoimmune applications.

Protective Effects of Oleic Acid Against Palmitic Acid-Induced Apoptosis in Pancreatic AR42J Cells and Its Mechanisms

Palmitic acid (PAM), one of the most common saturated fatty acid (SFA) in animals and plants, has been shown to induce apoptosis in exocrine pancreatic AR42J cells. In this study, we investigated cellular mechanisms underlying protective effects of oleic acid (OLA) against the lipotoxic actions of PAM in AR42J cells. Exposure of cells to long-chain SFA induced apoptotic cell death determined by MTT cell viability assay and Hoechst staining. Co-treatment of OLA with PAM markedly protected cells against PAM-induced apoptosis. OLA significantly attenuated the PAM-induced increase in the levels of pro-apoptotic Bak protein, cleaved forms of apoptotic proteins (caspase-3, PARP). On the contrary, OLA restored the decreased levels of anti-apoptotic Bcl-2 family proteins (Bcl-2, Bcl-xL, and Mcl-1) in PAM-treated cells. OLA also induced up-regulation of the mRNA expression of Dgat2 and Cpt1 genes which are involved in triacylglycerol (TAG) synthesis and mitochondrial beta-oxidation, respectively. Intracellular TAG accumulation was increased by OLA supplementation in accordance with enhanced expression of Dgat2 gene. These results indicate that restoration of anti-apoptotic/pro-apoptotic protein balance from apoptosis toward cell survival is involved in the cytoprotective effects of OLA against PAM-induced apoptosis in pancreatic AR42J cells. In addition, OLA-induced increase in TAG accumulation and up-regulation of Dgat2 and Cpt1 gene expressions may be possibly associated in part with the ability of OLA to protect cells from deleterious actions of PAM.

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...

MyD88 expression and anti-apoptotic signals of paclitaxel in epithelial ovarian cancer cells / 대한산부인과학회지

OBJECTIVE: The objectives of this study was to evaluate the correlation between myeloid differentiation protein 88 (MyD88) expression and paclitaxel effects on epithelial ovarian cancer cells and to evaluate whether paclitaxel had anti-apoptotic signals. METHODS: Epithelial ovarian cancer cells isolated from ascites and established cell lines were treated with increasing concentrations of paclitaxel (0.2 to 20 microM) for 24 and 48 hours and cell viability was determined using the CellTiter 96 AQueous One Solution Cell Proliferation Assay. Cytokine profiling was performed from culture supernatants using the Luminex 200 system. Nuclear factor-kappaB (NF-kappaB) activity was determined using a Luciferase reporter system. Levels of phospho-extracellular signal-regulated kinase (p-ERK) were measured by Western blot analysis. RESULTS: A strong signal for MyD88 expression was observed in R182, 01-19b and SKOV3 cells (MyD88-positive). A2780, R454 and 01-28 cells showed low levels of MyD88 (MyD88-negative). Paclitaxel effectively decreased cell viability in MyD88-negative A2780, R454, 01-28 cells after 24 and 48 hours (57%, 49%, 42% and 35%, 28%, 29%, respectively). MyD88-positive cells were resistant to paclitaxel. There was a significant increase in caspase-3/7 activity following paclitaxel treatment in MyD88-negative cells. No significant change in caspase-3/7 activity was detected in MyD88-positive cells. Paclitaxel induced NF-kappaB activation and enhanced the secretion of interleukin-6 (IL-6) and IL-8 in a dose dependent manner and induced ERK phosphorylation on MyD88-positive cells. CONCLUSION: Paclitaxel treatment for MyD88-positive ovarian cancer could have detrimental effects due to the paclitaxel-induced enhancement of NF-kappaB, ERK activities and pro-inflammatory cytokine production, which promote chemoresistance and tumor progression.

An Anti-apoptotic Protein Expressed Specifically and Highly in The Cardiac Tissue:ARC / 生物化学与生物物理进展

Many apoptosis-related heart diseases such as myocardial infarction, cardiomyopathies, and heart failure severely impair human's health and life. Currently, a key focus for the medical researchers is to find out effective ways to prevent or treat these heart diseases. ARC (apoptosis repressor with caspase recruitment domain) is the first anti-apoptotic protein so far identified to be highly and specifically expressed in the cardiac tissue. ARC could be structurally phosphorylated and involved in various signaling pathways during apoptosis.

Effect and underlying mechanism of antioxidant quercetin in mouse and in NIH-3T3 cells / 基础医学与临床

Objective To compare the difference in quercetin against oxidative stress response in mouse and in NIH-3T3 cells before and after H2O2 treatment,to explore the underlying mechanism for the quercetin antioxidant.Methods The cultured NIH-3T3 cells were randomly divided into 4 groups: quercetin(Q) pre-protective group(Qb) firstly treated with quercetin for 24 h followed by incubation with H2O2 for 30 min;post-protective group(Qa) treated with H2O2 for 30 min followed by incubation with quercetin for 24 h;H2O2 group(H2O2) after exposure to H2O2 for 30 min,incubated with DMEM medium and the control group(C) only cultured with DMEM medium.The survival rate and apoptotic rate were detected respectively with MTT and TUNEL in NIH-3T3 cell sus-pension samples.The expression of cyclin D1,PTEN,NF-?B,HSP-70,BCl-2,BAX and caspase-3 were examined with immunocytochemistry and immunoblotting.Besides,20 Wistar rats were divided into control group and experimental group,the latter was given with quercetin in the doze of 0.13 mmol/kg.The levels of T-AOC,SOD,GSH-Px,GSH,MDA,NOS and NO2-/NO3-were detected both in the cleaved NIH-3T3 cells and in the plasma from both experimental and control animals prior to and post-1 h,2 h and after 24 h.Results When the Qb group was compared with H2O2 or Qa group,the survival rate was higher and the apoptotic rate was lower.When the H2O2 group was compared with C group,the expression of cyclin D1、PTEN or BCl-2 was down-regulated;while that of BAX、HSP-70、NF-?B or caspase-3 was up-regulated;the level of T-AOC,SOD,GSH-Px or GSH was decreased;that of NOS、NO2-/NO3-or MDA enhanced in the cleft NIH-3T3 cells.When the plasma level of the anti-oxidative enzyme system prior to-compared with post-1h and 2h-treatment with Q,the level of T-AOC,SOD,GSH-Px and GSH,especially the former two,were higher;MDA,lower;NOS or NO2-/NO3-promoted.However,the above parameters basically became normal 24 h after treatment with Q.Conclusion Quercetin down-regulates the promoted expression of HSP70,NOS,NO2-/NO3-and NF-?B etc.in H2O2-treatment NIH-3T3 cells.Qb could reverse the H2O2 damage effects more markedly.Moreover,the quercetin exerts anti-oxidant protective effect through modulating the anti-oxidative enzyme system both in vivo and in vitro.However,based on the cell heterogeneity in none-or pre/post-H2O2-treatment state,a difference in quercetin antioxidant response is noted.

Tumor chemosensitization strategies based on apoptosis manipulations / 中国药理学通报

MDR(multidrug-resistance)is a major obstacle in the chemotherapy of cancer.Numerous mechanisms are known to contribute to MDR,alterations at the level of apoptosis control are one of those mechanisms except overexpression of drug efflux pumps.This review focuses on the research progression of alterations at the level of apoptosis inducing MDR,and some of the strategies that have been used in an attempt to chemosensitize resistant tumors by manipulating dysregulated apoptosis pathways.

Expression and significance of pro-apoptotic factors and anti-apoptotic factors in cervical intraepithelial neoplasia and cervical carcinoma / 第三军医大学学报

0.05).But the expression of Caspase-9 in AC group was significantly higher than that in CIN and squamous cell carcinoma group(P

Quantitation of the Early Apoptotic Cells Using Flow Cytometry / 대한임상병리학회지

BACKGROUND: There is a strong evidence that administration of anti-tumor drugs triggers apoptotic death of target cells. Therefore, quantitation of the 0early apoptotic cells could provide a very useful information for clinicians planning anti-tumor strategies. Therefore, we measured the amount of early apoptotic cells using annexin-FITC/PI dual fuorescence method by which early changes of apoptotic cell membrane could be detected. We also measured DNA content of apoptotic cells by PI single stain and performed DNA fragment assay simultaneously. METHODS: HL-60 cell line were cultured under 100, 200 ng/mL adriamycin for 12, 24, 36, 48 hours. Quantitation of the early apoptotic cells was done using flow cytometry with annexin-FITC and Propidium iodide (PI) dual fluorescence stain. And DNA content of the HL-60 cells was measured using PI single stain after fixing the cells. DNA ladder assay was also performed by agarose gel electrophoresis. RESULTS: The early apoptotic cells were 40.5% at adriamycin 100 ng/mL, after 24 hours culture and the secondary necrotic cells were 94.7% at adriamycin 200 ng/mL after 48 hours culture. There was a good correlation between annexin-PI stain and DNA content analysis. We could find DNA fragmentation on agarose gel electrophoresis. CONCLUSION: Quantitation of the early apoptotic cells using flow cytometry with annexin-PI dual fluorochrome stain and the DNA content analysis with PI single stain could be a good parameter for anti-apoptotic strategies.