|Year : 2013 | Volume
| Issue : 2 | Page : 80-83
Smac/DIABLO gene expression in acute myeloid leukemia patients
Ghada A. Suliman1, Maaly M. Mabrouk1, Enaam S. Rabee1, Amr Gawaly2
1 Department of Clinical Pathology, Faculty of Medicine, Tanta University, Tanta, Egypt
2 Department of Internal Medicine, Faculty of Medicine, Tanta University, Tanta, Egypt
|Date of Submission||14-Jan-2013|
|Date of Acceptance||14-Feb-2013|
|Date of Web Publication||20-Jun-2014|
Maaly M. Mabrouk
MD, Department of Clinical Pathology, Faculty of Medicine, Tanta University, 31527 Tanta
Source of Support: None, Conflict of Interest: None
Smac/DIABLO enhances apoptosis by antagonizing the inhibitors of apoptotic proteins. The expression of Smac/DIABLO in different cancers has been reported. The study aimed to evaluate Smac/DIABLO gene expression in patients with acute myeloid leukemia (AML) and also to determine its relation to the clinical outcome and survival of these patients.
Materials and methods
Smac/DIABLO gene expression was studied using real-time PCR in bone marrow samples from 70 newly diagnosed AML patients.
The Smac/DIABLO gene was expressed in 88.5% of AML patients. A total of 32 patients (51.6%) with positive Smac/DIABLO expression responded to treatment, and the remaining 30 patients (48.4%) were treatment resistant. Smac/DIABLO expression was associated with decreased lactate dehydrogenase levels and increased disease-free survival (P=0.01 and P<0.001, respectively). The expression was associated with an increase in the survival rate (P<0.05).
There was an increase of Smac/DIABLO expression in AML patients, and this was associated with the treatment response, increased disease-free survival, and better overall survival.
Keywords: acute myeloid leukemia, proapoptotic protein, Smac/DIABLO
|How to cite this article:|
Suliman GA, Mabrouk MM, Rabee ES, Gawaly A. Smac/DIABLO gene expression in acute myeloid leukemia patients. Egypt J Haematol 2013;38:80-3
|How to cite this URL:|
Suliman GA, Mabrouk MM, Rabee ES, Gawaly A. Smac/DIABLO gene expression in acute myeloid leukemia patients. Egypt J Haematol [serial online] 2013 [cited 2020 Jan 24];38:80-3. Available from: http://www.ehj.eg.net/text.asp?2013/38/2/80/134793
| Introduction|| |
Acute myeloid leukemia (AML) is a heterogeneous disease considering both clinical and genetic approaches 1. It occurs as a result of accumulation of genetic alterations in hematopoietic progenitor cells, disturbing their differentiation 2.
Several structural and numerical genetic aberrations have been encountered in AML, representing the independent prognostic factors for complete remission (CR), relapse risk, and overall survival (OS) 3,4.
Apoptosis, programmed cell death, is a genetically regulated biological process essential for normal tissue homeostasis 5. Evasion of apoptosis with more proliferative capacity and defective arrest are needed for malignant cell development 6.
Apoptosis is restarted by a group of cysteine–aspartic acid-specific proteases known as caspases 7. Caspases cause the morphological and the biochemical changes that associate with apoptosis 8. Activities of caspases are inhibited by a group of proteins called the inhibitor of apoptosis proteins (IAPs). This group of inhibitors includes XIAP, cIAP1, cIAP2, NAIP, livin, and survivin 9.
A mitochondria-derived proapoptotic protein named the second mitochondria-derived activator of caspases (Smac/DIABLO) has been identified. Smac/DIABLO functions by neutralizing the caspase-inhibitory effect of IAP proteins, specifically XIAP, which is the most potent caspase inhibitor 10.
The Smac peptide induces apoptosis provoked by chemotherapeutic or immunotherapeutic agents in several tumor cell lines 11. Smac/DIABLO expression should be studied to predict apoptotic changes in tumor cells in vivo 12.
This study was designed to evaluate Smac/DIABLO gene expression in AML patients and also to determine its relation to the clinical outcome and survival of these patients.
| Materials and methods|| |
A total of 70 patients with de-novo AML were selected from the Hematology Unit of the Department of Internal Medicine of Tanta University Hospitals (Egypt). They were treated and followed up in this study during August 2009 to August 2012. This study also included 10 apparently healthy individuals matched for age and sex with the patient group who were subjected to bone marrow (BM) aspiration for the exclusion of malignancies and had morphologically normal BM aspirates as the control group. Informed consent was obtained from all the studied groups. No karyotypic features were included in the study because some patients were missed for the karyotype analysis at diagnosis.
Clinical end points
CR was defined as: the recovery of morphologically normal BM aspirates and blood counts (i.e. absolute neutrophil count≥1.0×109 /l and platelet count≥100×109 /l), and no circulating leukemic blasts or evidence of extramedullary leukemia; a BM aspirate revealing normal maturation of all the cellular components (i.e. the erythrocytic, granulocytic, and megakaryocytic series); and presence of less than 5% of blast cells in the BM. A relapse was defined by the presence of 5% or more of BM blasts, circulating leukemic blasts, or development of extramedullary leukemia. The OS was measured from the date of diagnosis until the date of death, censoring for patients alive at the last follow-up. Disease-free survival (DFS) was measured from the date of CR until the date of relapse or death, regardless of the cause, censoring for patients alive at the last follow-up 13.
A complete blood count was performed using a fully automated blood cell counter (PCE-210; Erma Inc., Tokyo, Japan). Levels of lactate dehydrogenase (LDH) (kinetic method; supplied by DiaSys Diagnostic Systems GmbH, Holzheim, Germany) were also determined.
BM samples were collected at the time of diagnosis in EDTA tubes for flow cytometric immunophenotyping and gene analysis.
Flow cytometric immunophenotyping
Immunophenotyping of leukemic cells was performed on the BM samples using flow cytometry. A routine panel of monoclonal antibodies CD45, CD34, HLA-DR, CD3, CD7, CD10, CD19, CD79b, CD13, CD33, CD117, CD64, CD14, glycophorin A, CD61, and CD41 was used. The monoclonal antibodies were conjugated with fluorescein isothiocyanate and phycoerythrin (BD, FACS, Calibur; BD Pharmingen, San Diego, California, USA).
Smac/DIABLO gene expression
The gene expression was analyzed using a relatively quantitative real-time RT-PCR. The mononuclear cells were enriched using a Ficoll-Hypaque gradient, and isolation of total RNA was performed using a QIAamp RNA Blood Mini Kit (Qiagen GmbH, Hilden, Germany). All the RNA samples had an optical density 260/280 ratio ranging from 1.81 to 1.875, which confirms the good quality of the RNA. RNA samples with an optical density 260/280 ratio of less than 1.8 were excluded from the study.
For synthesis of single-stranded complementary DNA (cDNA), 10 μl of total cellular RNA was reverse transcribed into cDNA using the high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, California, USA), according to the manufacturer’s instructions. The synthesized cDNA samples were stored at −20°C until use.
Preparing the real-time PCR master mix for each sample
The cDNA is used as a template to amplify the studied genes and is normalized using glyceraldehydes-3-phosphate dehydrogenase (GAPDH) as a housekeeping gene. The assay identification numbers of target and housekeeping genes are as follow: Hs00219876_m1 (Smac/DIABLO), and GAPDH gene Hs99999905_m1 using the primers and probes assays listed in TaqMan gene expression assays from Applied Biosystems.
The reaction mixture was made up to a final volume of 25 μl and contained 5 μl of cDNA; 1.25 μl of assay mix [forward primer, reverse primer, and TaqMan probe mix (FAM dye), target gene (Smac/DIABLO)]; 1.25 μl [forward primer, reverse primer, TaqMan probe (Vic dye)] mix of reference gene (GAPDH); 12.5 µl of TaqMan Universal PCR Master Mix (Applied Biosystems), which consists of AmpliTaq Gold DNA polymerase, AmpErase UNG, dNTPs with dUTP, a Passive Reference ROX dye for PCR analysis, and an optimized buffer; and 5 μl of nuclease-free water. Real-time PCR was carried out with the following thermal profile: an initial denaturation step for 10 min at 95°C, followed by 40 cycles of annealing and elongation for 15 s at 95°C, and for 1 min at 60°C (Real-Time PCR Step One Instrument and Software; Applied Biosystems).
Instrumental raw data (fluorescence) of all the samples were converted into threshold cycles (Ct) using the SDS 1.2 software (Applied Biosystem). Ct values were then imported into an Excel worksheet for relative quantification (RQ).
For the calculation of RQ, the geometrical mean of GAPDH levels was used as a normalization factor.
The results were calculated by applying the comparative CT method for RQ, 2-ΔΔCt
The difference in the threshold cycles for the target and reference gene.
where ΔCTcalibrator is the mean value of control health human.
The fold change, which is defined as the ratio between the averaged normalized expression level of targets in the neoplastic and corresponding non-neoplastic samples, was calculated. Normalized RQ were log 2 transformed for statistical analysis.
The relative expression level of Smac/DIABLO mRNA was found to be significantly upregulated, up to 14-folds, in AML patients compared with healthy controls.
The normalized Smac/DIABLO mRNA level in AML patients was 14.7 (0.9–19.4), whereas in healthy controls it was 1.01 (0.3–2.2).
Continuous data were expressed as mean±SD and categorical data were expressed in number and percentages. Comparison of categorical data was made using the χ2-test. Comparison of continuous data between the two groups was made using Student’s t-test. Pearson’s correlation was used to determine the correlation between the different parameters. The survival charts were depicted on Kaplan–Meier plots. The expression levels of the Smac/DIABLO gene in the BM samples were categorized into high-expressor or low-expressor groups, depending on whether the target level was above or below the median expression value, respectively. Statistical significance was defined as a P-value of less than 0.05. Statistical presentation and analysis of the present study were carried out using the SPSS program version 17 (SPSS Inc., Chicago, Illinois, USA) and the Graph Pad Prism software (GraphPad Prism Software Inc., San Diego, California, USA).
| Results|| |
The clinical and laboratory data of the patients are presented in [Table 1].
Smac/DIABLO was highly expressed in 62 (88.5%) AML patients and had low expression in eight (11.5%) patients.
Of the patients, 32 (51.6%) with high Smac/DIABLO expression responded to treatment and the remaining 30 (48.4%) were treatment resistant. Seven patients with low Smac/DIABLO expression were treatment resistant. High Smac/DIABLO expression was associated with a good response to the treatment (χ2=4.2, P=0.04).
High Smac/DIABLO expression was associated with decreased LDH levels and increased DFS (P=0.01 and P<0.001, respectively) [Table 2].
The correlation analysis found negative correlations between DFS and WBC count and percentage of blasts in the peripheral blood (P=0.035 and 0.028, respectively) [Table 3].
|Table 3: Correlation of disease-free survival to the different parameters|
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The Kaplan–Meier curve revealed that Smac/DIABLO expression was associated with a better OS (long rank=5.9, P=0.014) [Figure 1].
|Figure 1: The Kaplan–Meier curve of Smac/DIABLO expression with the overall survival.|
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| Discussion|| |
During the last 30 years, there have been great advances in understanding the pathogenesis of AML to predict the disease outcome. AML is considered a heterogeneous disease based on the molecular and genetic levels that have been associated with pretreatment features and clinical outcomes of AML patients 1.
Smac/DIABLO has a proapoptotic effect that is mediated by its interaction with the IAPs and the release of caspases 9. Overexpression of Smac/DIABLO not only sensitizes neoplastic cells to apoptotic death 14,15 but also has a role during the initiation or progression of cancer 16–18.
Several studies approved that there is an increased expression of Smac/DIABLO in a wide variety of tumors, and this expression was associated with progression of metastasis or response to therapy, for example, in renal cell carcinoma 19, lung cancer 16, testicular germ cell tumors 20, hepatocellular carcinoma 21, and gastric adenocarcinoma 18.
Aveic et al. 22 reported that in their study, in the AML cell line, an enhanced expression of Smac/DIABLO was observed. In the present study, Smac/DIABLO was expressed 14-fold higher in AML patients compared with the control group. It was highly expressed in 88.5% of AML patients, and the high expression was associated with decreased LDH levels. High expression of the Smac/DIABLO gene was associated with a good response to treatment.
High expression of the Smac/DIABLO gene, decreased total leukocytic count, and decreased blast percentages in peripheral blood were associated with increased DFS and OS.
Pluta et al. 23 reported that there was an increase of Smac/DIABLO protein expression (96%) in newly diagnosed AML patients using flow cytometry. In their study, the decrease expression and unfavorable cytogenetics were associated with treatment failure. Furthermore, better OS was influenced by having an age of less than 60 years, percentage of BM blasts of less than 50% of cells, and the high expression of Smac/DIABLO. They also observed that the high expression of the Smac/DIABLO gene is an independent prognostic factor for a higher CR rate and longer OS in AML patients 23.
The cells with higher Smac/DIABLO expression and, therefore, lower apoptotic resistance should be more responsive to treatment and lesser to cancer progression; this should be helpful in making a decision for a different treatment strategy.
There was an increase of Smac/DIABLO expression in AML patients, and this was associated with the treatment response and increased DFS. Similar studies should be carried out on a large scale.
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[Table 1], [Table 2], [Table 3]