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Guibing R, Xiping Z, Xiaowen D, Dehong Z, Hongjiang Y, Xiaoru M, Wenju M, Xiangming H, Shuai Z. EFFECTS OF SALVIA MILTIORRHIZAE ON THE KIDNEY OF RATS WITH SEVERE ACUTE PANCREATITIS AND OBSTRUTIVE JAUNDICE. AFRICAN JOURNAL OF TRADITIONAL, COMPLEMENTARY, AND ALTERNATIVE MEDICINES : AJTCAM 2017; 14:103-124. [PMID: 28573227 PMCID: PMC5446434 DOI: 10.21010/ajtcam.v14i2.12] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 01/15/2023]
Abstract
Background: Severe acute pancreatitis (SAP) and obstructive jaundice (OJ) are frequent recurring diseases that bring about huge threat to human health. Some reports have demonstrated that Salviae miltiorrhizae can protect multiple organs of SAP and OJ model animals or patients, but their related mechanisms were not clear. In this study, we observed the effects of Salvia miltiorrhizae injection on apoptosis and NF-κB expression in kidney and explored the protective effect and mechanism of Salvia miltiorrhizae on the kidney of SAP or OJ rats. The results obtained will provide a theoretical basis for clinical application of Salvia miltiorrhizae. Material and Methods: A total of 288 rats were used for SAP -and OJ-associated experiments. The mortality rates of rats, the contents of serum BUN and CREA, the expression levels of Bax, NF-κB proteins and the apoptosis index were observed, respectively. Results: The pathological changes in the kidney of SAP or OJ rats in treated group were mitigated to varying degrees. At 6 and 12 hours after operation in SAP rats or on 21 and 28 days after operation in OJ rats, the contents of serum CREA in treated group were significantly lower than those in model control group; At 3 and 6 hours after operation, the staining intensity of Bax protein of kidney in treated group was significantly lower than that in model control group; on 14 days after operation, the apoptosis index in the kidney of OJ rats in treated group was significantly lower than that in model control group. Conclusion: Salvia miltiorrhizae can exert protective effects on the kidney of SAP and OJ rats.
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Affiliation(s)
- Ren Guibing
- Department of Oncological Surgery, Affiliated Hospital, Logistics University of the Chinese People's Armed Police Force, Tianjin, 300162, PR China
| | - Zhang Xiping
- Wenzhou Medical University, Wenzhou, 325035, Zhejiang Province, PR China.,Department of Breast Surgery, Zhejiang Cancer Hospital, Hangzhou 310022, Zhejiang Province, PR China
| | - Ding Xiaowen
- Department of Breast Surgery, Zhejiang Cancer Hospital, Hangzhou 310022, Zhejiang Province, PR China
| | - Zou Dehong
- Department of Breast Surgery, Zhejiang Cancer Hospital, Hangzhou 310022, Zhejiang Province, PR China
| | - Yang Hongjiang
- Department of Breast Surgery, Zhejiang Cancer Hospital, Hangzhou 310022, Zhejiang Province, PR China
| | - Meng Xiaoru
- Department of Oncological Surgery, Affiliated Hospital, Logistics University of the Chinese People's Armed Police Force, Tianjin, 300162, PR China
| | - Mo Wenju
- Department of Breast Surgery, Zhejiang Cancer Hospital, Hangzhou 310022, Zhejiang Province, PR China
| | - He Xiangming
- Department of Breast Surgery, Zhejiang Cancer Hospital, Hangzhou 310022, Zhejiang Province, PR China
| | - Zhao Shuai
- Department of Breast Surgery, Zhejiang Cancer Hospital, Hangzhou 310022, Zhejiang Province, PR China
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Baraka A, Shorbagy SE, Elfarargy OM, Haggag R, Abdelaziz LA, Elsayed SF, Elbana KA. Prognostic Significance of Apoptosis Regulators in B-Cell Chronic Lymphocytic Leukemia. ACTA ACUST UNITED AC 2017. [DOI: 10.4236/jct.2017.84032] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
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Al-Harbi S, Choudhary GS, Ebron JS, Hill BT, Vivekanathan N, Ting AH, Radivoyevitch T, Smith MR, Shukla GC, Almasan A. miR-377-dependent BCL-xL regulation drives chemotherapeutic resistance in B-cell lymphoid malignancies. Mol Cancer 2015; 14:185. [PMID: 26537004 PMCID: PMC4632834 DOI: 10.1186/s12943-015-0460-8] [Citation(s) in RCA: 37] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/29/2015] [Accepted: 10/20/2015] [Indexed: 01/15/2023] Open
Abstract
Background BCL-xL is an anti-apoptotic BCL-2 family protein that inhibits apoptosis and is overexpressed in many cancers. We have reported that acquired resistance to the BCL-2 inhibitor ABT-199 (venetoclax) is associated with increased BCL-xL expression. Yet, how BCL-xL mediates chemoresistance in hematopoietic malignancies is not clear. This finding may help in design of new strategies for therapeutic intervention to overcome acquired chemoresistance mediated by BCL-xL. Results We now show that the increased BCL-xL expression was inversely correlated with that of miR-377 in ABT-199-resistant cells. This finding was also extended to a panel of B-cell lymphoid lines and primary chronic lymphocytic leukemia (CLL) cells. miR-377 suppressed BCL-xL expression by recognizing two binding sites in the BCL-xL 3’-UTR. Mutation of these two miR-377 consensus-binding sites completely abolished its regulatory effect. Expression of a miR-377 mimic downregulated BCL-xL protein expression and significantly increased apoptotic cell death. Expression of a miR-377 inhibitor restored BCL-xL protein expression and limited cell death caused by the hypomethylating agent 5-azacytidine. Thus, miR-377-dependent BCL-xL regulation drives acquired therapeutic resistance to ABT-199. We further show that CLL patients who received a diverse array of chemotherapy regimens also had significantly higher BCL-xL and lower miR377 expression, indicating that exposure to chemotherapy might trigger transcriptional silencing of miR-377, which results in high levels of BCL-xL. Importantly, CLL patients with high BCL-xL/low miR-377 expression had an advanced tumor stage. Moreover, the high BCL-xL expression correlated with short treatment-free survival in 76 CLL patients. miR-377 is located at 14q32 in the DLK1-DIO3 region, which encodes the largest tumor suppressor miRNA cluster in humans. Examination of five additional 14q32 miRNAs revealed that the majority were significantly down-regulated in most CLL patients as well as in ABT-199-resistant cell lines. Remarkably, four of these miRNAs had significantly decreased expression in chemotherapy-treated CLL patients as compared to those untreated. These findings indicate a reduced expression of multiple miRNAs that may reflect a global silencing of this miRNA cluster in therapy-resistant lymphoid cells. Conclusions These findings reveal a novel mechanism by which down-regulation of miR-377 increases BCL-xL expression, promoting chemotherapy resistance in B-cell lymphoid malignancies. Electronic supplementary material The online version of this article (doi:10.1186/s12943-015-0460-8) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Sayer Al-Harbi
- Departments of Cancer Biology, Cleveland, OH, 44195, USA.,Department of Human Cancer Genomic Research, King Faisal Specialist Hospital and Research Cancer, Riyadh, 11211, Saudi Arabia
| | - Gaurav S Choudhary
- Departments of Cancer Biology, Cleveland, OH, 44195, USA.,Department of Pathology, Case Western Reserve University School of Medicine, Cleveland, OH, 44106, USA
| | - Jey Sabith Ebron
- Department of Biological, Geological, and Environmental Sciences, Center for Gene Regulation in Health and Disease, Cleveland State University, Cleveland, OH, 44115, USA
| | - Brian T Hill
- Department of Hematology and Oncology, Cleveland Clinic, Taussig Cancer Institute, Cleveland, OH, 44195, USA
| | | | - Angela H Ting
- Genomic Medicine Institute, Cleveland, OH, 44195, USA
| | - Tomas Radivoyevitch
- Quantitative Health Sciences, Lerner Research Institute, Cleveland, OH, 44195, USA
| | - Mitchell R Smith
- Department of Hematology and Oncology, Cleveland Clinic, Taussig Cancer Institute, Cleveland, OH, 44195, USA
| | - Girish C Shukla
- Department of Biological, Geological, and Environmental Sciences, Center for Gene Regulation in Health and Disease, Cleveland State University, Cleveland, OH, 44115, USA
| | - Alex Almasan
- Departments of Cancer Biology, Cleveland, OH, 44195, USA.
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Galluzzi L, Vitale I, Vacchelli E, Kroemer G. Cell death signaling and anticancer therapy. Front Oncol 2011; 1:5. [PMID: 22655227 PMCID: PMC3356092 DOI: 10.3389/fonc.2011.00005] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/18/2011] [Accepted: 04/21/2011] [Indexed: 12/22/2022] Open
Abstract
For a long time, it was commonly believed that efficient anticancer regimens would either trigger the apoptotic demise of tumor cells or induce a permanent arrest in the G1 phase of the cell cycle, i.e., senescence. The recent discovery that necrosis can occur in a regulated fashion and the increasingly more precise characterization of the underlying molecular mechanisms have raised great interest, as non-apoptotic pathways might be instrumental to circumvent the resistance of cancer cells to conventional, pro-apoptotic therapeutic regimens. Moreover, it has been shown that some anticancer regimens engage lethal signaling cascades that can ignite multiple oncosuppressive mechanisms, including apoptosis, necrosis, and senescence. Among these signaling pathways is mitotic catastrophe, whose role as a bona fide cell death mechanism has recently been reconsidered. Thus, anticancer regimens get ever more sophisticated, and often distinct strategies are combined to maximize efficacy and minimize side effects. In this review, we will discuss the importance of apoptosis, necrosis, and mitotic catastrophe in the response of tumor cells to the most common clinically employed and experimental anticancer agents.
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Cramer P, Hallek M. Prognostic factors in chronic lymphocytic leukemia-what do we need to know? Nat Rev Clin Oncol 2010; 8:38-47. [PMID: 20956983 DOI: 10.1038/nrclinonc.2010.167] [Citation(s) in RCA: 126] [Impact Index Per Article: 8.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
Of all leukemias, chronic lymphocytic leukemia (CLL) shows the highest variability in its clinical presentation and course. CLL can present as an aggressive and life threatening leukemia or as an indolent form that will not require treatment over decades. The currently available clinical staging systems for CLL are simple and inexpensive but lack accuracy to predict disease progression and survival on an individual basis. The increased understanding of the key events of molecular pathogenesis has provided a plethora of novel molecular and biological factors that correlate with the outcome of CLL. This Review provides a concise discussion of the most important discoveries and gives guidance on how to implement novel prognostic tools in the clinical management of CLL by applying the criteria of evidence, relevance, and simplicity to the selection of prognostic markers.
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Affiliation(s)
- Paula Cramer
- Department of Internal Medicine I, Center for Integrated Oncology Köln-Bonn, University of Cologne, Germany
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Bandyopadhyay S, Mitra R, Maulik U, Zhang MQ. Development of the human cancer microRNA network. SILENCE 2010; 1:6. [PMID: 20226080 PMCID: PMC2835996 DOI: 10.1186/1758-907x-1-6] [Citation(s) in RCA: 181] [Impact Index Per Article: 12.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 05/18/2009] [Accepted: 02/02/2010] [Indexed: 12/15/2022]
Abstract
BACKGROUND MicroRNAs are a class of small noncoding RNAs that are abnormally expressed in different cancer cells. Molecular signature of miRNAs in different malignancies suggests that these are not only actively involved in the pathogenesis of human cancer but also have a significant role in patients survival. The differential expression patterns of specific miRNAs in a specific cancer tissue type have been reported in hundreds of research articles. However limited attempt has been made to collate this multitude of information and obtain a global perspective of miRNA dysregulation in multiple cancer types. RESULTS In this article a cancer-miRNA network is developed by mining the literature of experimentally verified cancer-miRNA relationships. This network throws up several new and interesting biological insights which were not evident in individual experiments, but become evident when studied in the global perspective. From the network a number of cancer-miRNA modules have been identified based on a computational approach to mine associations between cancer types and miRNAs. The modules that are generated based on these association are found to have a number of common predicted target onco/tumor suppressor genes. This suggests a combinatorial effect of the module associated miRNAs on target gene regulation in selective cancer tissues or cell lines. Moreover, neighboring miRNAs (group of miRNAs that are located within 50 kb of genomic location) of these modules show similar dysregulation patterns suggesting common regulatory pathway. Besides this, neighboring miRNAs may also show a similar dysregulation patterns (differentially coexpressed) in the cancer tissues. In this study, we found that in 67% of the cancer types have at least two neighboring miRNAs showing downregulation which is statistically significant (P < 10-7, Randomization test). A similar result is obtained for the neighboring miRNAs showing upregulation in specific cancer type. These results elucidate the fact that the neighboring miRNAs might be differentially coexpressed in cancer tissues as that of the normal tissue types. Additionally, cancer-miRNA network efficiently detect hub miRNAs dysregulated in many cancer types and identify cancer specific miRNAs. Depending on the expression patterns, it is possible to identify those hubs that have strong oncogenic or tumor suppressor characteristics. CONCLUSIONS Limited work has been done towards revealing the fact that a number of miRNAs can control commonly altered regulatory pathways. However, this becomes immediately evident by accompanying the analysis of cancer-miRNA relationships in the proposed network model. These raise many unaddressed issues in miRNA research that have never been reported previously. These observations are expected to have an intense implication in cancer and may be useful for further research.
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Affiliation(s)
| | - Ramkrishna Mitra
- Machine Intelligence Unit, Indian Statistical Institute, Kolkata, India
| | - Ujjwal Maulik
- Department of Computer Science and Engineering, Jadavpur University, Kolkata, India
| | - Michael Q Zhang
- Watson School of Biological Sciences, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA
- MOE Key Laboratory of Bioinformatics and Bioinformatics Division, TNLIST, Tsinghua University, Beijing 100084, China
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Nückel H, Frey UH, Bau M, Sellmann L, Stanelle J, Dürig J, Jöckel KH, Dührsen U, Siffert W. Association of a novel regulatory polymorphism (−938C>A) in the BCL2 gene promoter with disease progression and survival in chronic lymphocytic leukemia. Blood 2006; 109:290-7. [PMID: 16960146 DOI: 10.1182/blood-2006-03-007567] [Citation(s) in RCA: 88] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Abstract
Bcl-2 plays a key role in the regulation of apoptosis. We investigated the role of a novel regulatory single-nucleotide polymorphism (−938C>A) in the inhibitory P2 BCL2 promoter in B-cell chronic lymphocytic leukemia (B-CLL). The −938C allele displayed significantly increased BCL2 promoter activity and binding of nuclear proteins compared with the A allele. Concomitantly, Bcl-2 protein expression in B cells from CLL patients carrying the −938 AA genotype was significantly increased compared with CC genotypes. Genotype distribution between 123 CLL patients (42 AA, 55 AC, 26 CC) and 120 genotyped healthy controls (36 AA, 63 AC, 21 CC) was not significantly different, suggesting that genotypes of this polymorphism do not increase the susceptibility for B-CLL. However, median time from first diagnosis to initiation of chemotherapy and median overall survival were significantly shorter in patients with −938AA genotype (38 and 199 months, respectively) compared with AC/CC genotypes (120 and 321 months, respectively; P = .008 and P = .003, respectively). Multivariable Cox regression identified the BCL2−938AA genotype as an independent prognostic factor for the time to first treatment (hazard ratio [HR] 1.9; P = .034) together with disease stage at diagnosis (HR 2.5; P = .004) and ZAP-70 status (HR 3.0; P = .001). The BCL2−938AA genotype is associated with increased Bcl-2 expression and a novel unfavorable genetic marker in patients with B-CLL.
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MESH Headings
- Aged
- Alleles
- Antineoplastic Combined Chemotherapy Protocols/therapeutic use
- Biomarkers, Tumor/genetics
- Cell Line
- Chromosome Aberrations
- Chromosomes, Human/genetics
- Chromosomes, Human/ultrastructure
- Disease Progression
- Female
- Genes, bcl-2
- Genotype
- Humans
- Kaplan-Meier Estimate
- Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy
- Leukemia, Lymphocytic, Chronic, B-Cell/genetics
- Leukemia, Lymphocytic, Chronic, B-Cell/mortality
- Male
- Middle Aged
- Neoplasm Proteins/genetics
- Polymorphism, Single Nucleotide
- Prognosis
- Promoter Regions, Genetic/genetics
- Proportional Hazards Models
- Retrospective Studies
- Survival Analysis
- Transcription, Genetic
- Transfection
- ZAP-70 Protein-Tyrosine Kinase/analysis
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Affiliation(s)
- Holger Nückel
- Department of Hematology, Medical Faculty, University of Duisburg-Essen, Germany.
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Kojima K, Konopleva M, McQueen T, O'Brien S, Plunkett W, Andreeff M. Mdm2 inhibitor Nutlin-3a induces p53-mediated apoptosis by transcription-dependent and transcription-independent mechanisms and may overcome Atm-mediated resistance to fludarabine in chronic lymphocytic leukemia. Blood 2006; 108:993-1000. [PMID: 16543464 PMCID: PMC1895860 DOI: 10.1182/blood-2005-12-5148] [Citation(s) in RCA: 186] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023] Open
Abstract
Although TP53 mutations are rare in B-cell chronic lymphocytic leukemia (CLL), Mdm2 overexpression has been reported as an alternative cause of p53 dysfunction. We investigated the potential therapeutic use of nongenotoxic p53 activation by a small-molecule antagonist of Mdm2, Nutlin-3a, in CLL. Nutlin-3a induced significant apoptosis in 30 (91%) of 33 samples from previously untreated patients with CLL; all resistant samples had TP53 mutations. Low levels of Atm (ataxia telangiectasia mutated) or high levels of Mdm2 (murine double minute 2) did not prevent Nutlin-3a from inducing apoptosis. Nutlin-3a used transcription-dependent and transcription-independent pathways to induce p53-mediated apoptosis. Predominant activation of the transcription-independent pathway induced more pronounced apoptosis than that of the transcription-dependent pathway, suggesting that activation of the transcription-independent pathway is sufficient to initiate p53-mediated apoptosis in CLL. Combination treatment of Nutlin-3a and fludarabine synergistically increased p53 levels, and induced conformational change of Bax and apoptosis in wild-type p53 cells but not in cells with mutant p53. The synergistic apoptotic effect was maintained in samples with low Atm that were fludarabine resistant. Results suggest that the nongenotoxic activation of p53 by targeting the Mdm2-p53 interaction provides a novel therapeutic strategy for CLL.
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MESH Headings
- Apoptosis/drug effects
- Ataxia Telangiectasia Mutated Proteins
- Cell Cycle Proteins/physiology
- Cells, Cultured
- DNA-Binding Proteins/physiology
- Drug Resistance, Neoplasm
- Drug Synergism
- Drug Therapy, Combination
- Humans
- Imidazoles/pharmacology
- Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy
- Leukemia, Lymphocytic, Chronic, B-Cell/pathology
- Mutation
- Piperazines/pharmacology
- Protein Serine-Threonine Kinases/physiology
- Proto-Oncogene Proteins c-mdm2/antagonists & inhibitors
- Transcription, Genetic
- Tumor Suppressor Protein p53/genetics
- Tumor Suppressor Protein p53/physiology
- Tumor Suppressor Proteins/physiology
- Vidarabine/analogs & derivatives
- Vidarabine/pharmacology
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Affiliation(s)
- Kensuke Kojima
- Department of Blood and Marrow Transplantation, Section of Molecular Hematology and Therapy, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd, Houston, 77030, USA
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Austen B, Powell JE, Alvi A, Edwards I, Hooper L, Starczynski J, Taylor AMR, Fegan C, Moss P, Stankovic T. Mutations in the ATM gene lead to impaired overall and treatment-free survival that is independent of IGVH mutation status in patients with B-CLL. Blood 2005; 106:3175-82. [PMID: 16014569 DOI: 10.1182/blood-2004-11-4516] [Citation(s) in RCA: 165] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
AbstractThe ataxia telangiectasia mutated (ATM) protein is the principal activator of the p53 protein in the response to DNA double-strand breaks. Mutations in the ATM gene have been previously found in B-cell chronic lymphocytic leukemias (B-CLLs) but their clinical significance is unknown. We analyzed 155 CLL tumors and found 12% with ATM mutations and 4% with TP53 mutations; 2 tumors contained mutations in both genes. Retrospective analysis on selected samples indicated that the ATM mutations were usually present at diagnosis. Compared with patients with wild-type ATM/TP53 genes, patients with ATM mutations had statistically significantly reduced overall and treatment-free survival. Although present in both IGVH mutation subgroups, ATM mutations were associated with unmutated IGVH genes and they provided independent prognostic information on multivariate analysis. Mutations in the ATM gene resulted in impaired in vitro DNA damage responses. Tumors with ATM mutations only partially correlated with tumors with loss of an ATM allele through an 11q deletion and, interestingly, those 11q-deleted tumors with a second wild-type ATM allele had a preserved DNA damage response. The majority of patients with ATM mutations were refractory to DNA damaging chemotherapeutic drugs and as such might benefit from therapies that bypass the ATM/p53 pathway.
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Affiliation(s)
- Belinda Austen
- Cancer Research United Kingdom (CRUK) Institute for Cancer Studies, University of Birmingham, Edgbaston, Birmingham, B15 2TT, United Kingdom.
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Brajusković G, Orolicki SV, Cerović S, Usaj SK, Marjanović S, Romac S. [Bcl-2 and Bax protein interaction in B-lymphocytes of peripheral blood in patients with chronic lymphocytic leukemia]. VOJNOSANIT PREGL 2005; 62:357-63. [PMID: 15913039 DOI: 10.2298/vsp0505357b] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/31/2023] Open
Abstract
BACKGROUND Chronic lymphocytic leukemia (CLL) is a neoplastic disease characterized by the accumulation of morphologically mature monoclonal CD5+ B cells in the early phase (G0/G1) of the cell cycle. The accumulation of neoplastically transformed B-lymphocytes (CLL cells) is primarily the consequence of apoptosis blocking in these cells. Bcl-2 proteins are well-known modulators of this process. Some of these proteins are anti-apoptotic while the others are pro-apoptotic. All contain at least one of the four conserved regions called the Bcl-2 homologous domains (BH1-BH4). Evidence indicates that Bcl-2 and Bax form homo- and heterodimers. The anti-apoptotic effect of Bcl-2 protein is based on its ability to bind Bax protein in the heterodimer form, and thus to block the forming of Bax/Bax proapoptotic homodimers. The ratio of Bcl-2/Bax represents the cell autonomous rheostat which determinates the type of the cell reaction to an apoptotic stimulus. METHODS The aim of this study was to determine the level of interaction between these two proteins in CLL cells using the co-immunoprecipition method. The study included the analysis of 20 peripheral blood specimens from 20 patients with CLL, and 20 peripheral blood specimens from healthy persons, who were in the control group. Specimens were precipitated with the monoclonal antibody for Bcl-2 protein, and immunoblotted with the palyclonal antibady for Bax protein (IP: Bcl-2/WB:Bax). At the same time, specimens were precipitated with the polyclonal antibody for Bax protein, and immunoblotted with the monoclonal antibody for Bcl-2 protein (IP: Bax/WB:Bcl-2). The intensity of Bcl-2 and Bax protein's binding compared to the control samples of the peripheral blood from healthy persons, was increased in CLL cells. RESULTS IP: Bax/WB: Bcl-2 showed a high level of "free" Bcl-2 protein which was not bound in the heterodimer form to Bax protein. Simultaneously IP: Bcl-2/WB: Bax showed that a higher quantity of Bax protein was bound in the heterodimer form to Bcl-2 protein as opposed to the quantity of pro-apoptotic Bax protein potentialy bound in the homodimer form. CONCLUSION Further studies involving larger groups of patients are necessary to explore the potential significance of the Bcl-2/Bax protein ratio as a prognositc parameter in the CLL treatment.
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Affiliation(s)
- Goran Brajusković
- ZPSM - Institut za patologiju, Crnotravska 17, 11040 Beograd, Srbija i Cma Gora.
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Zheng JY, Yang GS, Wang WZ, Li J, Li KZ, Guan WX, Wang WL. Overexpression of Bax induces apoptosis and enhances drug sensitivity of hepatocellular cancer-9204 cells. World J Gastroenterol 2005; 11:3498-503. [PMID: 15962362 PMCID: PMC4315948 DOI: 10.3748/wjg.v11.i23.3498] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the role of overexpression of Bax in apoptotic pathways and the response of human hepatocellular cancer (HCC)-9204 cells to cell death induced by adriamycin.
METHODS: The whole length of Bax cDNA was transfected into human HCC-9204 cells by the method of lipofectamine transfection. An inducible MT-II regulatory system was constructed, which allowed controlled expression of protein upon addition of ZnSO4 (100 μmol/L) as an external inducer. Stable transfecting inducible expression vector containing Bax gene was performed. Expression of Bax in protein was analyzed by immunohistochemistry and Western blotting. TUNEL and flow cytometry were used to assess the effect of Bax on apoptosis. Colony assay and tetrazolium blue (MTT) assay were used to evaluate the difference in drug sensitivity of HCC-9204 cells after Bax-transfection.
RESULTS: Immunohistochemistry and Western blotting demonstrated that the expression of Bax protein markedly increased in Bax-transfected cells 4 h after the addition of ZnSO4. Bax positive signal was frequently found on the cytoplasm and perinuclear region of HCC-9404 cells, and there was ectopic expression in cells with marked condensation of chromatin and cytoplasm (apoptotic cells). Apoptotic index significantly increased in Bax-transfected HCC-9204/Bax cells (3.6 vs 27.2, 4.2 vs 32.3, P<0.05). Flow cytometry analysis showed a significant sub-G1 peak and apoptosis in 15.4% HCC-9204/Bax cells 24 h after treatment. Furthermore, colony survival rate decreased from 66% (HCC-9204/pMD) to 45% (HCC-9204/Bax) 2 d after ADR withdrawal. MTT assay result showed that the effects of Bax on cell viability following ADR exposure were significant as compared to the vehicle-transfected HCC-9204/pMD cells (21% vs 44%, P<0.01).
CONCLUSION: Overexpression of Bax not only induces apoptosis, but also sensitizes HCC-9204 cells to cell death induced by adriamycin.
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Affiliation(s)
- Jian-Yong Zheng
- Department of Laparoscope, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai 200438, China
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12
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Kim JW, Tsukishiro T, Johnson JT, Whiteside TL. Expression of pro- and antiapoptotic proteins in circulating CD8+ T cells of patients with squamous cell carcinoma of the head and neck. Clin Cancer Res 2005; 10:5101-10. [PMID: 15297413 DOI: 10.1158/1078-0432.ccr-04-0309] [Citation(s) in RCA: 40] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
OBJECTIVE Apoptosis of T lymphocytes in the circulation of patients with squamous cell carcinoma of the head and neck (SCCHN) was shown to target effector CD8+ rather than CD4+ T cells. This study evaluates the contribution of pro- and antiapoptotic components of the mitochondria-dependent pathway to apoptosis of circulating CD8+ T cells in these patients. EXPERIMENTAL DESIGN Blood samples were obtained from 77 patients with SCCHN and 51 normal control(s) (NC). Percentages of CD8+Annexin V+ (ANX+) and CD8+CD95+ cells, changes in mitochondrial membrane potential and levels of expression of Bcl-2, Bcl-XL, and Bax in CD8+ T lymphocytes were measured by quantitative flow cytometry. RESULTS Elevated percentages (P < 0.001) of early apo-ptotic (CD8+ANX+ CD95+) T cells in the circulation distinguish SCCHN patients from NCs but not patients with no evidence of disease (NED) from those with active disease (AD). Circulating CD8+ but not CD4+ T cells in patients were found to contain higher levels of proapoptotic Bax and antiapoptotic Bcl-XL (P < 0.01) than NC cells. The Bax/Bcl-2 ratio was elevated in CD8+ T cells of patients relative to NCs (P < 0.01), and it correlated with the percentage of ANX+CD8+ T cells (P = 0.007). The Bax/Bcl-XL ratio discriminated AD from NED patients. CONCLUSION Apoptosis of circulating CD8+T cells is found in SCCHN patients with AD or NED. Up-regulated Bax and Bcl-XL expression, the elevated Bax/Bcl-2 ratio and its association with ANX binding implicate the mitochondrial pathway in death of CD8+ T cells of patients with SCCHN. Understanding of molecular mechanisms of T-cell death and survival is essential for the development of more effective biotherapies for SCCHN.
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Affiliation(s)
- Jeong-Whun Kim
- University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213, USA
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Saxena A, Viswanathan S, Moshynska O, Tandon P, Sankaran K, Sheridan DP. Mcl-1 and Bcl-2/Bax ratio are associated with treatment response but not with Rai stage in B-cell chronic lymphocytic leukemia. Am J Hematol 2004; 75:22-33. [PMID: 14695629 DOI: 10.1002/ajh.10453] [Citation(s) in RCA: 97] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/07/2022]
Abstract
Although both Bcl-2/Bax ratio and Mcl-1 have been identified to be of clinical relevance in B-cell chronic lymphocytic leukemia (CLL), there is controversy regarding their role; further, their relative importance is not well delineated. Expression of Bcl-2, Bax, the Bcl-2/Bax ratio, and Mcl-1 in 51 consecutive previously untreated CLL patients and 16 controls was determined by Western blotting. Only 37 patients were treated, all with chlorambucil and prednisone initially. Six patients achieved complete response (CR), 14 were non-responders (NR), and 17 had a partial response (PR), as defined by NCI criteria. There was considerable inter-patient variability in protein expression and overlap with healthy volunteers (P > 0.05). All patients with CR had low Mcl-1 levels compared to the PR + NR group (0.07 +/- 0.02 vs. 0.14 +/- 0.07, P = 0.043). Higher Mcl-1 expression as determined by dichotomizing the data was associated with a failure to achieve CR (P = 0.021). The Bcl-2/Bax ratio was significantly associated with treatment response only when CR and PR were considered together (0.89 +/- 0.53 [CR + PR] vs. 3.38 +/- 4.47 [NR], P = 0.0118). There was no association with Rai stage. Low Mcl-1 appears to be a requirement for CR, while low Bcl-2/Bax ratio is indicative of some response to conventional treatment.
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MESH Headings
- Aged
- Aged, 80 and over
- Antineoplastic Agents, Alkylating/therapeutic use
- Antineoplastic Agents, Hormonal/therapeutic use
- Antineoplastic Combined Chemotherapy Protocols/therapeutic use
- Chlorambucil/therapeutic use
- Female
- Humans
- Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy
- Leukemia, Lymphocytic, Chronic, B-Cell/metabolism
- Leukemia, Lymphocytic, Chronic, B-Cell/pathology
- Male
- Middle Aged
- Myeloid Cell Leukemia Sequence 1 Protein
- Neoplasm Proteins/metabolism
- Neoplasm Staging
- Prednisone/therapeutic use
- Proto-Oncogene Proteins/metabolism
- Proto-Oncogene Proteins c-bcl-2/metabolism
- ROC Curve
- Treatment Outcome
- bcl-2-Associated X Protein
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Affiliation(s)
- Anurag Saxena
- Department of Pathology, Royal University Hospital and College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
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Viatour P, Bentires-Alj M, Chariot A, Deregowski V, de Leval L, Merville MP, Bours V. NF- kappa B2/p100 induces Bcl-2 expression. Leukemia 2003; 17:1349-56. [PMID: 12835724 DOI: 10.1038/sj.leu.2402982] [Citation(s) in RCA: 127] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
The NF-kappaB2/p100 and bcl-3 genes are involved in chromosomal translocations described in chronic lymphocytic leukemias (CLL) and non-Hodgkin's lymphomas, and nuclear factor kappaB (NF-kappaB) protects cancer cells against apoptosis. Therefore, we investigated whether this transcription factor could modulate the expression of the Bcl-2 antiapoptotic protein. Bcl-2 promoter analysis showed multiple putative NF-kappaB binding sites. Transfection assays of bcl-2 promoter constructs in HCT116 cells showed that NF-kappaB can indeed transactivate bcl-2. We identified a kappaB site located at position -180 that can only be bound and transactivated by p50 or p52 homodimers. As p50 and p52 homodimers are devoid of any transactivating domains, we showed that they can transactivate the bcl-2 promoter through association with Bcl-3. We also observed that stable overexpression of p100 and its processed product p52 can induce endogenous Bcl-2 expression in MCF7AZ breast cancer cells. Finally, we demonstrated that, in breast cancer and leukemic cells (CLL), high NF-kappaB2/p100 expression was associated with high Bcl-2 expression. Our data suggest that Bcl-2 could be an in vivo target gene for NF-kappaB2/p100.
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Affiliation(s)
- P Viatour
- Center for Cellular and Molecular Therapy, University of Liège, Liège, Belgium
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Anether G, Tinhofer I, Senfter M, Greil R. Tetrocarcin-A--induced ER stress mediates apoptosis in B-CLL cells via a Bcl-2--independent pathway. Blood 2003; 101:4561-8. [PMID: 12560233 DOI: 10.1182/blood-2002-08-2501] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Tetrocarcin-A (TC-A), an antibiotic agent isolated from actinomycetes, has recently been described to antagonize Bcl-2 functions, thereby sensitizing tumor cells to cell death signals under control of Bcl-2. In this study, we analyzed the direct proapoptotic effect of TC-A in the B-chronic lymphocytic leukemia (B-CLL) model. We focused on the signal cascade triggered by TC-A in B-CLL cells and identified activated mitochondrial as well as endoplasmatic reticulum (ER) stress signals. The expression levels of known effector molecules mediating mitochondrial signaling, such as Bax and Bid, and the antagonistic molecule Bcl-2 did not influence sensitivity of B-CLL cells to TC-A. Furthermore, the molecular chaperone and sensor of ER stress, HSP70, though significantly up-regulated in B-CLL cells undergoing TC-A-triggered apoptosis, was ineffective to exert its anti-apoptotic function described in multiple cell death pathways. Autologous T cells of B-CLL patients were significantly less sensitive to TC-A as were also T cells from healthy donors when compared with their normal B-cell fraction. Furthermore, sensitivity of B-CLL cells to TC-A treatment in vitro was dependent neither on the expression levels of CD38-a prognostic factor for survival of B-CLL patients as well as for their response to therapy-nor on the clinical stage or pretreatment status of patients. From our data showing that TC-A induced a cell death pathway via ER stress preferentially in B cells and that it acted independently of important markers of drug sensitivity and of clinical markers, we conclude that TC-A might represent an attractive candidate drug for further evaluation in preclinical trials.
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Affiliation(s)
- Gabriele Anether
- Department of Internal Medicine, University of Innsbruck, Innsbruck, Austria
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Abstract
Flow cytometry has been extensively used to follow the apoptotic cascade and to enumerate apoptotic cells, both in cell cultures and, to a lesser extent, in tissue biopsies. An overview of the apoptotic cascade and how flow cytometric measurements can be used to observe the different elements of this process is presented.
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Suvas S, Singh V, Sahdev S, Vohra H, Agrewala JN. Distinct role of CD80 and CD86 in the regulation of the activation of B cell and B cell lymphoma. J Biol Chem 2002; 277:7766-75. [PMID: 11726649 DOI: 10.1074/jbc.m105902200] [Citation(s) in RCA: 178] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
To date, not much has been known regarding the role of CD80 and CD86 molecules in signaling of B cells. The CD28/CTLA4 ligands, CD80 (B7-1) and CD86 (B7-2), are expressed on the surface of freshly isolated splenic B cells, and their expression is up-regulated by lipopolysaccharides. In the present study, we have investigated whether signaling via CD80/CD86 could alter the proliferation and immunoglobulin synthesis of B cells. Splenic B cells were stimulated with lipopolysaccharides in the presence of anti-B7-1 (16-10A1) and anti-B7-2 (GL1) monoclonal antibodies (mAbs). Exciting features observed during the study were that cross-linking of CD86 with GL1 enhanced the proliferation and production of IgG1 and IgG2a isotypes. In contrast, anti-B7-1 (16-10A1) mAb could efficiently block the proliferation and production of IgG1 and IgG2a. Furthermore, GL1 mAb could also induce the secretion of IgG isotypes from B cell lymphomas. Importantly, 16-10A1 could retard the growth of lymphomas and favored the up-regulation of pro-apoptotic molecules caspase-3, caspase-8, Fas, FasL, Bak, and Bax and down-regulation of anti-apoptotic molecule Bcl-x(L). In contrast, GL1 augmented the level of anti-apoptotic molecules Bcl-w and Bcl-x(L) and decreased the levels of pro-apoptotic molecule caspase-8, thereby providing a novel insight into the mechanism whereby triggering through CD80 and CD86 could deliver regulatory signals. Thus, this study is the first demonstration of a distinct signaling event induced by CD80 and CD86 molecules in B cell lymphoma. Finally, the significance of the finding is that CD80 provided negative signal for the proliferation and IgG secretion of normal B cells and B cell lymphomas. In contrast, CD86 encouraged the activity of B cells.
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Affiliation(s)
- Susmit Suvas
- Immunology Laboratory, Institute of Microbial Technology, Postgraduate Institute of Medical Education & Research, Chandigarh 160036, India
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