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Additional file 1. Methodological Appendix.

Mahnaz Mohammadi Kian, 1, 2 Mahdieh Salemi, 1, 2 Mohammad Bahadoran, 3 Atousa Haghi, 1, 4 Nasrin Dashti, 5 Saeed Mohammadi, 1, 2 Shahrbano Rostami, 1, 2 Bahram Chahardouli, 1, 2 Davood Babakhani, 1 Mohsen Nikbakht 1, 2 1Hematology Oncology and Stem Cell Transplantation Research Center, Tehran University of Medical Sciences, Tehran, Iran; 2Hematologic Malignancies Research Center, Tehran University of Medical Sciences, Tehran, Iran; 3Department of Biochemistry, Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran; 4Young Researchers & Elite Club Tehran Medical Sciences, Islamic Azad University, Tehran, Iran; 5Department of Medical Laboratory Sciences, School of Allied Health Sciences, Tehran University of Medical Sciences, Tehran, IranCorrespondence: Mohsen NikbakhtHematology, Oncology and Stem Cell Transplantation Research Center, Tehran University of Medical Sciences, Tehran, IranTel +982184902639Fax +982188004140Email m-nikbakht@sina.tums.ac.irNasrin DashtiDepartment of Medical Laboratory Sciences, School of Allied Health Sciences, Tehran University of Medical Sciences, Tehran, IranTel +989123583690Email dashti@tums.ac.irIntroduction: Acute myeloid leukemia (AML) is a type of blood disorder that exhibits uncontrolled growth and reduced ability to undergo apoptosis. Signal transducer and activator of transcription 3 (STAT3) is a family member of transcription factors which promotes carcinogenesis in most human cancers. This effect on AML is accomplished through deregulation of several critical genes, such as B cell lymphoma-extra-large (BCL-XL) which is anti-apoptotic protein. The aim of this study was to evaluate the effect of curcumin (CUR) and thalidomide (THAL) on apoptosis induction and also the alteration of the mRNA expression level of STAT3 and BCL-XL mRNA on AML cell line compounds.Methods: The growth inhibitory effects of CUR and THAL and their combination were measured by MTT assay in U937 and KG-1 cell lines. The rates of apoptosis induction and cell cycle analysis were measured by concurrent staining with Annexin V and PI. The mRNA expression level of STAT3 and BCL-XL was evaluated by Real-Time PCR.Results: CUR inhibited proliferation and induced apoptosis in both KG-1 and U937 cells and this effect increased by combination with THAL. The expression level of STAT3 and BCL-XL was significantly down-regulated in KG-1 cells after treatment by CUR and THAL and their combination.Conclusion: Overall, our findings suggested that down-regulation of STAT3 and BCL-XL mRNA expression in response to CUR and THAL treatment lead to inhibition of cell growth and induction of apoptosis.Keywords: acute myeloid leukemia, curcumin, thalidomide, STAT3, Bcl-xL

Acute myeloid leukemia (AML) is a blood disorder characterized by uncontrolled proliferation of myeloid progenitors and decrease in the apoptosis rate. The vascular endothelial growth factor (VEGF) promotes blood vessel regeneration which might play important roles in development and progression of neoplasia. Our previous studies focused on cytotoxicity and anticancer effects of arsenic trioxide (ATO) and thalidomide (THAL) as an anti-VEGF compound in the AML cell model. ATO also affects regulatory genes involved in cell proliferation and apoptosis. The aim of present study was to examine the effects of ATO and THAL alone and in combination on U937 and KG-1 cells , with attention to mRNA expression for VEGF isoforms. Growth inhibitory effects was assessed by MTT assay and apoptosis induction was determined by Annexin/PI staining. mRNA expression levels were evaluated by real-time PCR. Our data indicated that ATO (1.618μM and 1μM in KG-1 and U937 cell lines respectively), THAL (80μM and 60μM) and their combination inhibited proliferation and induced apoptosis in our cell lines. mRNA expression of VEGF (A, B) decreased while C and D isoforms did not show any significant changes. Taken together, according to the obtained results, the VEGF autocrine loop could be a target as a therapeutic strategy for cases of AML.

Despite impressive advances in therapeutic approaches, long-term survival with acute myeloid leukemia (AML) is low as a result of treatment resistance and frequent relapse. Among multitude oncogenic proteins involved in the acquisition of a chemo-resistant phenotype, osteopontin (OPN) recently has attracted marked attention. In spite of the well-defined association between OPN expression and cure rate with solid tumors, there is a scarcity of information on any role of this protein in AML cases. Based on the critical role of OPN in cell survival, it seems reasonable to hypothesize that isoform expression levels may impact on the regulation of apoptosis in AML cells in response to conventional chemotherapeutic drugs and its relation to relapse. To investigate associations between induction of apoptosis and OPN isoform expression, two distinct AML cell lines (KG-1 as a leukemic stem cell model and U937) were treated with chemotherapy drugs, and cell viability and apoptosis were evaluated by MTT and Annexin/PI assay. After determination of appropriate drug doses, mRNA expression levels of OPN isoforms and OPN-related genes were investigated. Our results demonstrated for the first time that acquired up-regulation of OPN-b and c isoforms might prevent conventional chemotherapy regimen-induced apoptosis in AML cells. Moreover, upregulation of OPN-b and c in AML cells appears concurrent with upregulation of AKT/VEGF/CXCR4/STAT3/ IL-6 gene expression. To sum up, this study suggests that OPN-b and c isoforms could be considered as unique beneficial molecular biomarkers associated with leukemic stem cell chemoresistance. Hence, they have potential as molecular candidates for detection of minimal residual disease (MRD) and determination of remission in AML patients. Further evaluation with quantitative real time PCR on patient samples for confirmation appears warranted.

Background: Interfering with cell proliferation and survival is a critical role for antineoplastic drugs leading to cell death through induction of apoptosis. Alternative treatments with herbal extracts offer insights into acute myeloid leukemia (AML) therapy. Parthenolide (PTL), an extract from feverfew, induces apoptosis in primary human leukemia stem cells (LSCs) and bulk leukemic cell populations. Osteopontin (OPN) preserves cell viability in response to anticancer agents and its receptors could be utilized for therapeutic targeting of cancer cells. Methods: U937 cells were cultured in RPMI 1640 with concentrations of 2, 4, 6, 8, and 10 μM PTL for 20-24 hours for MTT assays. Apoptosis assays were performed with Annexin V-Alexa Fluor-488/PI as Annexin V+/PI- and Annexin V+/PI+ to measure early and late apoptosis, respectively. Quantitative real-time PCR was used to measure OPN gene expression using the 2-ΔΔCt method. The PTL–treated cells were stained with FITC-CD38 antibody for flow cytometry analyses. Data were compared using one-way analysis of variance (ANOVA) by SPSS 19. Results: Parthenolide inhibited growth of U937 cells with IC25 and IC50 values of 4 and 5.8 μM, respectively. Death induction with PTL was apoptotic. Flow cytometry showed a significant decrease in the percentage of CD38+ U937 cells in response to PTL. Osteopontin gene expression decreased in response to PTL. Conclusions: PTL induced apoptosis and reduced OPN gene expression in U937 cells.