Plasma-derived exosomal miR-326, a prognostic biomarker and novel candidate for treatment of drug resistant pediatric acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is a cancer with high incidence rate in pediatrics and drug resistance is a major clinical concern for ALL treatment. The current study was designed to evaluate the role of exosomal miR-326 in diagnosis and treatment of children with B-ALL. Exosomes were isolated from plasma samples of 30 patients and B-ALL cell lines followed by characterization, using nanoparticle tracking analysis, immunoblotting assay and electron microscopy. qPCR showed significantly increased levels of miR-326 in patients exosomes compared with non-cancer controls (P < 0.05, AUC = 0.7500). Moreover, a comparison between the sensitive and drug resistant patients revealed a prognostic value for the exosomal miR326 (P < 0.05, AUC = 0.7755). Co-culture studies on drug resistant patient primary cells and B-ALL cell lines suggested that exosomes with high miR-326 level act as vehicles for reducing cells viability. B-ALL cell line transfection with naked miR-326 mimic confirmed the results, and fluorescence microscopy validated uptake and internalization of exosomes by target cells. The novel introduced features of the exosomal miR-326 address a non-invasive way of diagnosing primary drug resistance in pediatric ALL and advocates a novel therapeutic strategy for this cancer.

GingerenoneA overcomes dexamethasone resistance by activating apoptosis and inhibiting cell proliferation in pediatric T-ALL cells.

Plant-based combination strategies have been widely considered in cancer therapy to attenuate chemotherapeutics side effects. The anti-leukemic effect of the whole ginger extract was previously portrayed by our team, and the current study is centered around the cytotoxicity and mechanism of action of a phenolic subsidiary of ginger, GingerenoneA, on pediatric acute lymphoblastic leukemia. GingernoneA imposed, dose-dependently, inhibitory effects on the viability of T and B leukemia cell lines confirmed by MTT assays. Resistance to Dexamethasone, a mostly used chemotherapeutic in acute lymphoblastic leukemia treatments, was overcome by GingernoneA. A synergistic effect of Dexamethasone and GingrenoneA on T leukemia cell lines and patient primary cells was confirmed. Annexin-V/PI and acridine orange/ethidium bromide staining illustrated dose-dependent apoptosis in CCRF-CEM cells developed by GingerenoneA. The intrinsic and extrinsic apoptosis induction and antiproliferative attribution of GingerenoneA were validated by western blot and qPCR. Despite the supposed loss of function in CCRF-CEM cells, TP53 showed increased expression levels and functional activity upon treatment with GingernoneA. Bioinformatic studies revealed the conceivable impact of GingerenoneA on the reactivity of mutant P53 through its binding to Cys124. Our findings may provide novel strategies for therapeutic intervention to ameliorate pALL outcomes.

The effects of Astragalus polysaccharides, tragacanthin, and bassorin on methotrexate-resistant acute lymphoblastic leukemia.

Background and purpose: One strategy to overcome methotrexate (MTX) resistance in acute lymphoblastic leukemia is suppressing MDR1 expression. It has been proved Astragalus polysaccharides (APS) exert their anticancer effect by reversing drug resistance. Due to the structural similarity of tragacanthin and bassorin with APS, we aimed to investigate the effects of the aforementioned polysaccharides on the expression of the MDR1 gene in the MTX-treated CCRF-CEM cells. Experimental approach: Cytotoxicity of APS, bassorin, and tragacanthin on CCRF-CEM, CCRF-CEM/MTX (cells treated with MTX at IC50), and CCRF-CEM/R cells (CCRF-CEM cells resistant to MTX) was evaluated by MTT assay. The effect of all three compounds on MDR1 expression was evaluated using RT-PCR. Findings/Results: All the concentrations of tragacanthin, bassorin, and APS (except at 0.8-100 µg/mL in CCRF-CEM) decreased the viability of all the cells compared to the negative control group; and against the positive control (MTX-treated cells), only bassorin at 20-100 µg/mL in CCRF-CEM/R and tragacanthin at 50 and 100 µg/mL in CCRF-CEM/MTX and at 2-100 µg/mL in CCRF-CEM/R decreased cell viability. Tragacanthin diminished MDR1 expression in CCRF-CEM/MTX and CCRF-CEM/R cells, which MTX had already induced. Conclusion and implication: According to the results of this study, tragacanthin was a potent cytotoxic agent against CCRF-CEM cells and enhanced the chemosensitivity of CCRF-CEM/MTX and CCRF-CEM/R cells to MTX by down-regulation of MDR1 gene expression. Therefore, it could be a promising compound against cancer. Other possible mechanisms of action of tragacanthin should be evaluated and further in vitro and in vivo investigations are required.

miR-16-5p enhances sensitivity to RG7388 through targeting PPM1D expression (WIP1) in Childhood Acute Lymphoblastic Leukemia.

Aim: Given the encouraging results of the p53-Mdm2 inhibitor RG7388 in clinical trials and the vital function of miR-16-5p in suppressing cell proliferation, the aim of the present study was to investigate the combined impact of RG7388 and miR-16-5p overexpression on the childhood acute lymphoblastic leukemia (chALL). Methods: miRTarBase and miRDB, along with KEGG and STRING databases, were used to predict miR-16-5p target genes and explore protein-protein interaction networks, respectively. B- and T-lymphoblastic cell lines, in addition to patient primary cells, were treated with RG7388. Ectopic overexpression of miR-16-5p in Nalm6 cell line was induced through cell electroporation and transfection of microRNA mimics was confirmed by qRT-PCR. Cell viability was evaluated using the MTT assay. Western blot analyses were performed to evaluate the effects of RG7388 and miR-16-5p upregulation on the protein levels of p53 and its downstream target genes in chALL cells. Paired sample t-test was employed for statistical analyses. Results: MTT assay showed RG7388-induced cytotoxicity in wild-type p53 Nalm6 cell line and p53 functional patient primary cells. However, CCRF-CEM and p53 non-functional leukemic cells indicated drug resistance. Western blot analyses validated the bioinformatics results, confirming the downregulation of WIP1, p53 stabilization, as well as overexpression of p21WAF1 and Mdm2 proteins in Nalm6 cells transfected with miR-16-5p. Moreover, enhanced sensitivity to RG7388 was observed in the transfected cells. Conclusion: This is the first study indicating the mechanistic importance of miR-16-5p overexpression in chALL and its inhibitory role in leukemia treatment when combined with the p53-Mdm2 antagonist, RG7388. These findings might be useful for researchers and clinicians to pave the way for better management of chALL.

Potential anti-cancer effects of hibernating common carp (Cyprinus carpio) plasma on B16-F10 murine melanoma: In vitro and in vivo studies.

Melanoma is the major type of skin cancer, which its treatment is still a challenge in the world. In recent years, interest in hibernation-based therapeutic approaches for various biomedical applications has been increased. Many studies indicated that some factors in the blood plasma of hibernating animals such as alpha-2-macroglobulin (A2M) cause anti-proliferative effects. Considering that, the present study was conducted to investigate the anti-cancer effects of hibernating common carp plasma (HCCP) on murine melanoma (B16-F10) in vitro and in vivo. The effect of HCCP on cell viability, migration, apoptosis rate, and cell cycle distribution of B16-F10 cells, tumor growth, and rate of survival were evaluated. To investigate the role of A2M in the anti-cancer effects of HCCP, the gene of interest and proteins in HCCP and non-hibernating common carp plasma (NHCCP) were evaluated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry analysis. Based on our findings, HCCP significantly decreased B16-F10 cell viability. Moreover, HCCP caused morphological alternations, inhibition of migration, induction of apoptosis, and significantly induced the cell cycle arrest at the G2/M phase. In addition, A2M level was significantly increased in HCCP compared with NHCCP. Taken together, our findings suggested that HCCP had the potential to be a promising novel therapeutic target for cancer treatment because of its anti-cancer properties.

Puzzling out iron complications in cancer drug resistance.

Iron metabolism are frequently disrupted in cancer. Patients with cancer are prone to anemia and receive transfusions frequently; the condition which results in iron overload, contributing to serious therapeutic complications. Iron is introduced as a carcinogen that may increase tumor growth. However, investigations regarding its impact on response to chemotherapy, particularly the induction of drug resistance are still limited. Here, iron contribution to cell signaling and various molecular mechanisms underlying iron-mediated drug resistance are described. A dual role of this vital element in cancer treatment is also addressed. On one hand, the need to administer iron chelators to surmount iron overload and improve the sensitivity of tumor cells to chemotherapy is discussed. On the other hand, the necessary application of iron as a therapeutic option by iron-oxide nanoparticles or ferroptosis inducers is explained. Authors hope that this paper can help unravel the clinical complications related to iron in cancer therapy.

6-Shogaol induces apoptosis in acute lymphoblastic leukaemia cells by targeting p53 signalling pathway and generation of reactive oxygen species.

Combination therapies, using medicinal herbs, are broadly recommended to attenuate the chemotherapy adverse effects. Based on our previous findings considering the anti-leukaemic effects of ginger extract on acute lymphoblastic leukaemia (ALL) cells, the present study was aimed to investigate the anti-cancer role of this pharmaceutical plant on ALL mice models. Moreover, we worked towards identifying the most anti-leukaemic derivative of ginger and the mechanism through which it may exert its cytotoxic impact. In vivo experiments were performed using five groups of six C57BL/6 nude mice, and the anti-leukaemic activity of ginger extract alone or in combination with methotrexate (MTX) was examined. Results showed increased survival rate and reduced damages in mice brain and liver tissues. Subsequently, MTT assay demonstrated synergistic growth inhibitory effect of 6-shogaol (6Sh) and MTX on ALL cell lines and patients primary cells. Eventually, the molecular anti-neoplastic mechanism of 6Sh was evaluated using Bioinformatics. Flow cytometry illustrated 6Sh-mediated apoptosis in Nalm-6 cells confirmed by Western blotting and RT-PCR assays. Further analyses exhibited the generation of reactive oxygen species (ROS) through 6Sh. The current study revealed the in vivo novel anti-leukaemic role of ginger extract, promoted by MTX. Moreover, 6-shogaol was introduced as the major player of ginger cytotoxicity through inducing p53 activity and ROS generation.

Increased level of long non coding RNA H19 is correlated with the downregulation of miR-326 and BCL-2 genes in pediatric acute lymphoblastic leukemia, a possible hallmark for leukemogenesis.

Long non-coding RNAs (lncRNAs) and their role in competitive endogenous RNA (ceRNA) networks have emerged as fundamental debates in the biological processes of initiation and progression of cancer. This study aimed to identify and measure the expression levels of relevant ceRNA regulatory genes contributing to acute lymphoblastic leukemia (ALL). lncRNA H19 and BCL-2 mRNA were chosen based on in silico studies and their interactions with miR-326. Subsequently, the aforementioned coding/non-coding gene expression profiles were measured using qRT-PCR in 50 bone marrow samples, including 33 cases with pediatric ALL and 17 controls with no evidence of malignancy. lncRNA H19 was identified as an oncogenic factor which was noticeably increased in the newly diagnosed patients (P = 0.0019, AUC = 0.84) and negatively associated with miR-326 (r = -0.6, P = 0.02). Furthermore, a negative correlation was introduced between the transcriptional levels of miR-326 and the anti-apoptotic BCL-2 gene (r = -0.6, P = 0.028). The novel experimental and bioinformatic results achieved in this study may provide new insights into the molecular leukemogenesis of pediatric ALL.

Selective dysregulation of ABC transporters in methotrexate-resistant leukemia T-cells can confer cross-resistance to cytarabine, vincristine and dexamethasone, but not doxorubicin.

Pediatric acute lymphoblastic leukemia (pALL) includes 75 % of childhood leukemias, and methotrexate (MTX) is one of the most effective chemotherapy agents prescribed for pALL treatment. The aim of this study was to establish and characterize an MTX-resistant tumor cell model in order to study the mechanism contributing to drug sensitivity loss in pALL. Parental CCRF-CEM cells were treated with a gradual increasing concentration of MTX from 5 nM to 1.28 µM. The resistant subline was then characterized according to the cellular morphology, cellular growth curves and specific mRNA expression changes associated with drug resistance in ALL. Moreover, in vitro cytotoxicity assays were used to analyze cells relative responsiveness to a set of clinically used anti-ALL chemotherapy drugs. The morphological changes observed in the new R-CCRF-CEM/MVCD subline were associated with dysregulation of the EMT-related genes, Twist1 and CDH1. Cells demonstrated downregulation of ABCC1 and the overexpression of ABCA2, ABCA3, and ABCB1 membrane transporters. However, short treatment of the sensitive and parental cell line with MTX did not affect the expression profiles of the former ABC pumps. Moreover, R-CCRF-CEM/MVCD cells demonstrated cross-resistance to cytarabine (cytosine arabinoside, ara-C), vincristine, and dexamethasone, but not doxorubicin. The induced cross-resistance to specific chemotherapy drugs may possibly be attributed to selective dysregulation of the ABC transporters and EMT-related genes. These data may pave the way for the development of new cancer therapeutic strategies.

Publisher Correction: Fatty acid synthase, a novel poor prognostic factor for acute lymphoblastic leukemia which can be targeted by ginger extract.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Fatty acid synthase, a novel poor prognostic factor for acute lymphoblastic leukemia which can be targeted by ginger extract.

Altered metabolism of fatty acid synthesis is considered a hallmark characteristic of several malignancies, including acute lymphoblastic leukemia (ALL). To evaluate the impact of fatty acid synthase (FASN) on drug resistant ALL, bone marrow samples were collected from 65 pediatric ALLs, including 40 de novo and 25 relapsed patients. 22 non-cancer individuals were chosen as controls. Quantitative RT-PCR showed increased expression levels of FASN in drug resistant patients compared with the therapy responders. Single and combined treatment of malignant cells were analyzed using Annexin-V/PI double staining and MTT assays. Incubation of resistant primary cells with ginger showed simultaneous increased apoptosis rates and reduced FASN expression levels. Furthermore, docking studies demonstrated high affinity bindings between ginger derivatives and FASN thioesterase and ketosynthase domains, compared with their known inhibitors, fenofibrate and morin, respectively. Finally, combined treatment of in-house multidrug resistant T-ALL subline with ginger and dexamethasone induced drug sensitivity and down regulation of FASN expression, accordingly. To the best of our knowledge, this is the first study that introduces FASN upregulation as a poor prognostic factor for drug resistant childhood ALL. Moreover, it was revealed that FASN inhibition may be applied by ginger phytochemicals and overcome dexamethasone resistance, subsequently.

Simultaneous changes in expression levels of BAALC and miR-326: a novel prognostic biomarker for childhood ALL.

OBJECTIVE: Multidrug resistance and consequent relapse are two major obstacles for treating children with acute lymphoblastic leukemia, the most frequent childhood malignancy. MicroRNAs have potential regulatory roles in response to chemotherapy. The goal of this study was to determine the microRNA that may have effects on the expression level of brain and acute lymphoblastic leukemia (BAALC) and to investigate the in vitro and ex vivo association between their expression levels. METHODS: In silico tools were utilized to determine a putative miRNA targeting BALLC. Quantitative real-time polymerase chain reaction was used to investigate expression levels of BAALC and its predicted microRNA, miR-326, in bone marrow samples of 30 children with acute lymphoblastic leukemia and 13 controls, in addition to the resistant and parental CCRF-CEM cell lines. To assess the status of response to therapy, minimal residual disease was measured using single-strand conformation polymorphism. RESULTS: MiR-326 was selected due to the strong possibility of its interaction with BAALC according to the obtained in silico results. Statistical analysis showed a significant downregulation of miR-326 and overexpression of BALLC in drug-resistant acute lymphoblastic leukemia cell line and patients compared with the parental cell line and drug-sensitive patients, respectively (P = 0.015, 0.005, 0.0484 and 0.0005, respectively). The expression profiles of miR-326 and BAALC were inversely correlated (P = 0.028). CONCLUSIONS: The results introduced the inversely combined expression levels of miR-326 and BAALC as a novel, independent prognostic biomarker for pediatric acute lymphoblastic leukemia (P = 0.007). Moreover, bioinformatics data showed a possible regulatory role for miR-326 on BAALC mRNA, which may possibly contribute to the development of drug resistance in patients with childhood acute lymphoblastic leukemia.

Sal-Like Protein 4 Transcription Factor: A Significant Diagnostic Biomarker Involved in Childhood ALL Resistance and Relapse.

PURPOSE: Sal-like protein 4 transcription factor (SALL4) is a stem cell transcription factor that plays an essential role in the maintenance and self-renewal of embryonic and hematopoietic stem cells, functioning as an oncogene in several cancers. However, the role of SALL4 in the biological behavior of childhood acute lymphoblastic leukemia and its relationship with multidrug resistance and relapse has remained largely unknown. PATIENTS AND METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) was used to characterize the expression pattern of SALL4 in the bone marrow samples of 43 patients with Philadelphia negative ALL and 18 children in the non-cancer control group. The presence of minimal residual disease was measured a year after the initial therapy using SSCP (single-strand conformation polymorphism). In addition, the correlation between the expression of SALL4 and ABCA3 in relapsed patients was analyzed statistically. RESULTS: Results showed an overexpression of SALL4 in de novo patients compared with the control group (P=0.0001, AUC= 0.93), indicating the importance of this gene in the induction of leukemia. A significant increase in the ABCA3 expression levels was revealed in the relapsed patients, in comparison with the drug-sensitive group (P = 0.0005). The leukemogenetic effect of SALL4 can be related to the effect of this gene on the maintenance of pluripotency in cancer stem cells. Results also suggest that the expression of SALL4 can be considered as a diagnostic marker for pediatric ALL. Moreover, SALL4 expression levels in the minimal residual disease positive (mrd+) ALL group was significantly higher than those in the mrd- group (p=0.0001, AUC= 0.92). CONCLUSION: These data demonstrate the prognostic impact of SALL4 in childhood ALL. Our findings also indicated a direct correlation between the mRNA expression levels of SALL4 and ABCA3 transporter in the relapsed group of ALL patients (r=0.7). These results describe a possible mechanism by which SALL4 may lead to the development of multidrug resistance.

Iron protects childhood acute lymphoblastic leukemia cells from methotrexate cytotoxicity.

Drug resistance is a fundamental clinical concern in pediatric acute lymphoblastic leukemia (pALL), and methotrexate (MTX) is an essential chemotherapy drug administered for the treatment. In the current study, the effect of iron in response to methotrexate and its underlying mechanisms were investigated in pALL cells. CCRF-CEM and Nalm6 cell lines were selected as T and B-ALL subtypes. Cells were pretreated with ferric ammonium citrate, exposed to the IC50 concentration of MTX and cell viability was assessed using MTT, colony formation, and flow cytometry assays. Iron-loaded cells were strongly resistant to MTX cytotoxicity. The inhibitory effect of N-acetyl cysteine to reverse the acquired MTX resistance was greater than that of the iron chelator, deferasirox, highlighting the importance of iron-mediated ROS in MTX resistance. Subsequently, the upregulation of BCL2, SOD2, NRF2, and MRP1 was confirmed using quantitative RT-PCR. Moreover, a positive correlation was demonstrated between the MRP1 expression levels and bone marrow iron storage in pALL patients. Further supporting our findings were the hematoxylin and eosin-stained histological sections showing that iron-treated nude mice xenografts demonstrated significantly more liver damage than those unexposed to iron. Overall, iron is introduced as a player with a novel role contributing to methotrexate resistance in pALL. Our findings suggest that the patients' bone marrow iron stores are necessary to be assessed during the chemotherapy, and transfusions should be carefully administrated.

Quercetin-Conjugated Superparamagnetic Iron Oxide Nanoparticles Protect AlCl3-Induced Neurotoxicity in a Rat Model of Alzheimer's Disease via Antioxidant Genes, APP Gene, and miRNA-101.

Alzheimer's disease (AD) is a neurodegenerative disease with cognitive impairment. Oxidative stress in neurons is considered as a reason for development of AD. Antioxidant agents such as quercetin slow down AD progression, but the usage of this flavonoid has limitations because of its low bioavailability. We hypothesized that quercetin-conjugated superparamagnetic iron oxide nanoparticles (QT-SPIONs) have a better neuroprotective effect on AD than free quercetin and regulates the antioxidant, apoptotic, and APP gene, and miRNA-101. In this study, male Wistar rats were subjected to AlCl3, AlCl3 + QT, AlCl3 + SPION, and AlCl3 + QT-SPION for 42 consecutive days. Behavioral tests and qPCR were used to evaluate the efficiency of treatments. Results of behavioral tests revealed that the intensity of cognitive impairment was decelerated at both the middle and end of the treatment period. The effect of QT-SPIONs on learning and memory deficits were closely similar to the control group. The increase in expression levels of APP gene and the decrease in mir101 led to the development of AD symptoms in rats treated with AlCl3 while these results were reversed in the AlCl3 + QT-SPIONs group. This group showed similar results with the control group. QT-SPION also decreased the expression levels of antioxidant enzymes along with increases in expression levels of anti-apoptotic genes. Accordingly, the antioxidant effect of QT-SPION inhibited progression of cognitive impairment via sustaining the balance of antioxidant enzymes in the hippocampus of AD model rats.

The antitoxic effects of quercetin and quercetin-conjugated iron oxide nanoparticles (QNPs) against H2O2-induced toxicity in PC12 cells.

BACKGROUND: We recently showed that quercetin-conjugated iron oxide nanoparticles (QNPs) promoted the bioavailability of quercetin (Qu) in the brain of rats and improved the learning and memory of diabetic rats. In this study, we characterized the modifications in the antitoxic effects of Qu after conjugation. MATERIALS AND METHODS: We conjugated Qu to dextran-coated iron oxide nanoparticles (DNPs) and characterized DNPs and QNPs using FTIR, XRD, DLS, Fe-SEM, and EDX analyzes. The antiradical properties of Qu, DNPs, and QNPs were compared by 2, 2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity assay. Catalase-like activities of DNPs and QNPs were estimated using catalase activity assay kit, and the antitoxic effects of Qu and QNPs were evaluated with spectrophotometry, MTT assay, flow cytometry, and real-time q-PCR. RESULTS: Qu had a stronger anti-radical activity than DNPs and its activity decreased after being conjugated to DNPs. The catalase-like activity of DNPs remained intact after conjugation. DNPs had less toxicity on PC12 cells viabilities as compared to free Qu, and the conjugation of Qu with DNPs attenuated its cytotoxicity. Furthermore, MTT assay results indicated 24 h pretreatment with Qu had more protective effects than QNPs against H2O2-induced cytotoxicity, while Qu and QNPs had the same effects for 48 and 72 h incubation. Although the total antioxidant capacity of Qu was attenuated after conjugation, the results of flow cytometry and real-time q-PCR confirmed that 24 h pretreatment with the low concentrations of Qu and QNPs had the similar antioxidant, anti-inflammatory, and anti-apoptotic effects against the cytotoxicity of H2O2. CONCLUSION: Qu and QNPs showed the similar protective activities against H2O2-induced toxicity in PC12 cells. Given the fact that QNPs have magnetic properties, they may serve as suitable carriers to be used in neural research and treatment.

The Hedgehog signal transducer Smoothened and microRNA-326: pathogenesis and regulation of drug resistance in pediatric B-cell acute lymphoblastic leukemia.

PURPOSE: Multidrug resistance (MDR) and the subsequent disease relapse are the major causes of childhood acute lymphoblastic leukemia (ALL) related death. The Hedgehog (Hh) signaling pathway can contribute to cancer MDR. In the current study, Smoothened (Smo) was selected as the experimental target due to its importance in the Hh pathway in order to evaluate its probable role in pediatric B-ALL drug resistance. PATIENTS AND METHODS: The study included 27 pediatric B-ALL and 16 control bone marrow samples. Quantitative RT-PCR was used to investigate the expression levels of Smo and miR-326 as the key players of the Hh pathway. Western blot analysis was performed. The presence of minimal residual disease was studied using PCR-SSCP. The association between Smo expression and drug resistance was analyzed statistically. RESULTS: Results showed a significant increase in the Smo expression levels in drug-resistant patients in comparison with drug-sensitive children with B-ALL (P=0.0128, AUC=0.82). A considerable negative association between miR-326 and Smo expression levels was identified (r=-0.624, P=0.002). A binomial test confirmed the regulatory role of miR-326 on the translational repression of Smo (P=0.031). Statistics showed no association between Smo and ABCA2 expression levels. However, a significant positive correlation was observed between the Smo and ABCA3 transcripts in the resistant ALL children (r=0.607, P=0.016). CONCLUSION: Data revealed the possible oncogenic impact of Smo on leukemogenesis and drug resistance in pediatric B-ALL. Upregulation of Smo was introduced, for the first time, as a prognostic factor for drug resistance in childhood B-ALL. To the best of our knowledge, this is the first study that shows a positive correlation between Smo and ABCA3 expression levels in pediatric B-ALL, explaining a possible mechanism for the development of drug resistance in this cancer. Moreover, the current project revealed a negative modulatory effect of miR-326 on the expression levels of Smo.

Ginger extract has anti-leukemia and anti-drug resistant effects on malignant cells.

PURPOSE: Based on the poor prognosis of drug resistance in pediatric acute lymphoblastic leukemia (ALL) and adverse effects of chemotherapy, this study was aimed to evaluate the effect of several herbal extracts on leukemic cells. METHODS: Two subtypes of T- and B-ALL cell lines, followed by ALL primary cells were treated with cinnamon, ginger, and green tea extracts, alone or in combination with methotrexate (MTX). Possible apoptosis was investigated using Annexin-V/PI double staining. Real-time PCR was applied to evaluate the expression levels of related ABC transporters upon combination therapy. RESULTS: The IC50s for cinnamon, ginger and green tea extracts on ALL cell lines were 300 µg/ml, 167 µg/ml and 70 µg/ml, respectively. Surprisingly, the methotrexate (MTX)-resistant sub-line showed more sensitivity to ginger. Combined treatment with ginger and MTX showed synergistic effects on CCRF-CEM, Nalm-6 and ALL primary cells. It was shown that ginger does not impair the high expression levels of ABCA2 or ABCA3 transporter genes in the ALL malignant cells, suggesting other molecular pathways involved in its anticancer potential. CONCLUSION: To the best of our knowledge, this is the first study that reveals the antileukemic effect of ginger extract on both, pediatric ALL cell lines and primary cells.

Targeting p53-MDM2 Interaction Using Small Molecule Inhibitors and the Challenges Needed to be Addressed.

MDM2 protein is the core negative regulator of p53 that maintains the cellular levels of p53 at a low level in normal cells. Mutation of the TP53 gene accounts for 50% of all human cancers. In the remaining malignancies with wild-type TP53, p53 function is inhibited through other mechanisms. Recently, synthetic small molecule inhibitors have been developed which target a small hydrophobic pocket on MDM2 to which p53 normally binds. Given that MDM2-p53 antagonists have been undergoing clinical trials for different types of cancer, this review illustrates different aspects of these new cancer targeted therapeutic agents with the focus on the major advances in the field. It emphasizes on the p53 function, regulation of p53, targeting of the p53-MDM2 interaction for cancer therapy, and p53-dependent and -independent effects of inhibition of p53-MDM2 interaction. Then, representatives of small molecule MDM2-p53 binding antagonists are introduced with a focus on those entered into clinical trials. Furthermore, the review discusses the gene signatures in order to predict sensitivity to MDM2 antagonists, potential side effects and the reasons for the observed hematotoxicity, mechanisms of resistance to these drugs, their evaluation as monotherapy or in combination with conventional chemotherapy or with other targeted therapeutic agents. Finally, it highlights the certainly intriguing questions and challenges which would be addressed in future studies.

Recovery of MicroRNA from Stored Bone Marrow Aspirate Slides.

BACKGROUND: Archived bone marrow aspirate slides are almost infinite, readily available resource of biospecimens that enable retrospective molecular investigations of diseases. RNAs obtained from slides has limitations in utility because of their low quality and highly fragmented nature. MicroRNAs are small (<22 nt) noncoding RNAs with various cellular regulatory roles. Due to their small size, microRNAs are less prone to degradation and modification, therefore, can be preserved well in archived tissues. METHODS: The current study investigated the efficacy of archived bone marrow aspirate slides for miRNA expression analysis in pediatric leukemia. Total RNA was isolated from air-dried unstained archived slides using High pure miRNA isolation Kit with some modifications and from fresh samples using TRizol. After cDNA synthesis, RT-qPCR was then carried out using specific hsa-miR-326 LNA primers. Finally, statistical analyses were conducted using GraphPad Prism 6 software. RESULTS: The difference observed in miRNA expression due to disease state was far greater than the differences between archived slides and their matching fresh bone marrow specimens. In fact, the expression of archival slide smears for the miR-326 closely mimicked that of fresh-frozen tissues (0.035±0.04 vs. 0.03±0.04) (Mean±SD, p>0.05). Differential expression of hsa-miR-326 was detected between leukemic and non-leukemic samples from archived slides or fresh frozen bone marrows. CONCLUSION: The demonstration that archived bone marrow aspirate slides can be utilized for miRNA expression studies offers tremendous potential for future investigations into the role that miRNAs play in the development and long term outcome of hematologic, as well as non-hematologic diseases.