The sensitivity of acute myeloid leukemia cells to cytarabine is increased by suppressing the expression of Heme oxygenase-1 and hypoxia-inducible factor 1-alpha.

BACKGROUND: Acute myeloid leukemia (AML), a malignancy Often resistant to common chemotherapy regimens (Cytarabine (Ara-c) + Daunorubicin (DNR)), is accompanied by frequent relapses. Many factors are involved in causing chemoresistance. Heme Oxygenase-1 (HO-1) and Hypoxia-Inducible Factor 1-alpha (HIF-1α) are two of the most well-known genes, reported to be overexpressed in AML and promote resistance against chemotherapy according to several studies. The main chemotherapy agent used for AML treatment is Ara-c. We hypothesized that simultaneous targeting of HO-1 and HIF-1α could sensitize AML cells to Ara-c. METHOD: In this study, we used our recently developed, Trans-Activator of Transcription (TAT) - Chitosan-Carboxymethyl Dextran (CCMD) - Poly Ethylene Glycol (PEG) - Nanoparticles (NPs), to deliver Ara-c along with siRNA molecules against the HO-1 and HIF-1α genes to AML primary cells (ex vivo) and cell lines including THP-1, KG-1, and HL-60 (in vitro). Subsequently, the effect of the single or combinational treatment on the growth, proliferation, apoptosis, and Reactive Oxygen Species (ROS) formation was evaluated. RESULTS: The designed NPs had a high potential in transfecting cells with siRNAs and drug. The results demonstrated that treatment of cells with Ara-c elevated the generation of ROS in the cells while decreasing the proliferation potential. Following the silencing of HO-1, the rate of apoptosis and ROS generation in response to Ara-c increased significantly. While proliferation and growth inhibition were considerably evident in HIF-1α-siRNA-transfected-AML cells compared to cells treated with free Ara-c. We found that the co-inhibition of genes could further sensitize AML cells to Ara-c treatment. CONCLUSIONS: As far as we are aware, this study is the first to simultaneously inhibit the HO-1 and HIF-1α genes in AML using NPs. It can be concluded that HO-1 causes chemoresistance by protecting cells from ROS damage. Whereas, HIF-1α mostly exerts prolific and direct anti-apoptotic effects. These findings imply that simultaneous inhibition of HO-1 and HIF-1α can overcome Ara-c resistance and help improve the prognosis of AML patients.

Targeted Silencing of NRF2 by rituximab-conjugated nanoparticles increases the sensitivity of chronic lymphoblastic leukemia cells to Cyclophosphamide.

BACKGROUND: Targeting influential factors in resistance to chemotherapy is one way to increase the effectiveness of chemotherapeutics. The nuclear factor erythroid 2-related factor 2 (Nrf2) pathway overexpresses in chronic lymphocytic leukemia (CLL) cells and appears to have a significant part in their survival and chemotherapy resistance. Here we produced novel nanoparticles (NPs) specific for CD20-expressing CLL cells with simultaneous anti-Nrf2 and cytotoxic properties. METHODS: Chitosan lactate (CL) was used to produce the primary NPs which were then respectively loaded with rituximab (RTX), anti-Nrf2 Small interfering RNA (siRNAs) and Cyclophosphamide (CP) to prepare the final version of the NPs (NP-Nrf2_siRNA-CP). All interventions were done on both peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (BMNCs). RESULTS: NP-Nrf2_siRNA-CP had satisfying physicochemical properties, showed controlled anti-Nrf2 siRNA/CP release, and were efficiently transfected into CLL primary cells (both PBMCs and BMNCs). NP-Nrf2_siRNA-CP were significantly capable of cell apoptosis induction and proliferation prevention marked by respectively decreased and increased anti-apoptotic and pro-apoptotic factors. Furthermore, use of anti-Nrf2 siRNA was corresponding to elevated sensitivity of CLL cells to CP. CONCLUSION: Our findings imply that the combination therapy of malignant CLL cells with RTX, CP and anti-Nrf2 siRNA is a novel and efficient therapeutic strategy that was capable of destroying malignant cells. Furthermore, the use of NPs as a multiple drug delivery method showed fulfilling properties; however, the need for further future studies is undeniable. Video Abstract.

The role of SF3B1 and NOTCH1 in the pathogenesis of leukemia.

The discovery of new genes/pathways improves our knowledge of cancer pathogenesis and presents novel potential therapeutic options. For instance, splicing factor 3b subunit 1 (SF3B1) and NOTCH1 genetic alterations have been identified at a high frequency in hematological malignancies, such as leukemia, and may be related to the prognosis of involved patients because they change the nature of malignancies in different ways like mediating therapeutic resistance; therefore, studying these gene/pathways is essential. This review aims to discuss SF3B1 and NOTCH1 roles in the pathogenesis of various types of leukemia and the therapeutic potential of targeting these genes or their mutations to provide a foundation for leukemia treatment.

The molecular biology and therapeutic potential of Nrf2 in leukemia.

NF-E2-related factor 2 (Nrf2) transcription factor has contradictory roles in cancer, which can act as a tumor suppressor or a proto-oncogene in different cell conditions (depending on the cell type and the conditions of the cell environment). Nrf2 pathway regulates several cellular processes, including signaling, energy metabolism, autophagy, inflammation, redox homeostasis, and antioxidant regulation. As a result, it plays a crucial role in cell survival. Conversely, Nrf2 protects cancerous cells from apoptosis and increases proliferation, angiogenesis, and metastasis. It promotes resistance to chemotherapy and radiotherapy in various solid tumors and hematological malignancies, so we want to elucidate the role of Nrf2 in cancer and the positive point of its targeting. Also, in the past few years, many studies have shown that Nrf2 protects cancer cells, especially leukemic cells, from the effects of chemotherapeutic drugs. The present paper summarizes these studies to scrutinize whether targeting Nrf2 combined with chemotherapy would be a therapeutic approach for leukemia treatment. Also, we discussed how Nrf2 and NF-κB work together to control the cellular redox pathway. The role of these two factors in inflammation (antagonistic) and leukemia (synergistic) is also summarized.

The prognostic and therapeutic potentials of CTLA-4 in hematological malignancies.

INTRODUCTION: Hematological Malignancies (HMs) are a group of progressive, difficult-to-treat, and highly recurrent diseases. A suppressed phenotype of the immune system is present in HMs and growing evidence indicates the role of Cytotoxic T lymphocyte-Associated protein 4 (CTLA-4) in the course of HMs. AREAS COVERED: This article reviews the recent literature on the role of CTLA-4 in different subtypes of HMs. Here, the studies on the expression pattern, its effect on the prognosis of different HMs, and polymorphisms of CTLA-4 have been elaborated. Finally, the effect of targeting CTLA-4 in vitro and in vivo, as well as in clinical trials, is discussed. EXPERT OPINION: According to the recent literature, CTLA-4 is overexpressed in different HMs, which is correlated with poor survival, while it is associated with better a prognosis in Chronic Lymphocytic Leukemia (CLL). Targeting CTLA-4 in Acute Myeloid Leukemia (AML), Sezary Syndrome (SS), Hodgkin's Lymphoma (HL), and so on, is helpful. While this is not recommended and may even be harmful in multiple myeloma (MM) and CLL. Also, it seems that certain CTLA-4 gene polymorphisms are efficient factors in the course of HMs. Future studies may broaden our knowledge regarding the role of CTLA-4 in HMs.

Enhanced Solubility and One-Step Purification of Functional Dimeric Carboxypeptidase G2.

Carboxypeptidase G2 is a bacterial enzyme that catalyzes methotrexate conversion to its inactive forms which are then eliminated via a non-renal pathway in patients with renal disorders during a high-dose methotrexate administration. Due to the increasing demand of this enzyme, it was of interest to simplify its production process. For this reason, we developed a method for production and one-step purification of this enzyme using an intein-mediated system with a chitin-binding affinity tag. The carboxypeptidase G2 gene from Pseudomonas RS16 was optimized, synthesized, cloned into the pTXB1 expression vector and finally transformed into Escherichia coli BL21 (DE3) cells. The optimal condition for the enzyme soluble expression was achieved in 2×YT medium containing 1% glucose at 25°C for 30 h with 0.5 mM IPTG. The enzyme without intein was expressed as inclusion bodies indicating the importance of intein for the protein solubility. The expressed homodimer protein was purified to homogeneity on a chitin affinity column. The Km and kcat values of 6.5 µM and 4.57 s-1, respectively, were obtained for the purified enzyme. Gel filtration analysis indicated that the resulting recombinant protein was a dimer of 83 kDa. Fluorescence and circular dichroism spectroscopy confirmed the enzyme tertiary and secondary structures, respectively. The use of intein-mediated system provided the possibility of the one-step carboxypeptidase G2 purification, paving the way to the application of this enzyme in pharmaceutics.

Rational design toward developing a more efficient laccase: Catalytic efficiency and selectivity.

Laccases are multicopper oxidases that catalyze the oxidation of variety of substrates. The specificity and efficiency of laccases are clearly the important components leading to their remarkable uses. To develop an improved biocatalysts, site directed mutagenesis of laccase from Bacillus HR03 was carried out in the current study. Based on the ABTS-bound crystal structure of CotA from B. subtilis and alignment with closely related enzymes, T415 and T418 at the vicinity of the type 1 copper site were chosen and several mutants (T415I, T418I, T415G, T415G/T418I) were made. Kinetic parameters of the constructs were then determined using ABTS and SGZ as substrates. In comparison with the wild-type, catalytic efficiency toward ABTS was improved by 4 fold in T415I and 1.5 fold in T418I and T415G. T415I and T418I variants were identified to be almost 11 and 27 times more specific for ABTS than for SGZ compared with the wild type. T415I was also found to acquire enhanced thermal stability with the half-life of 60min at 80°C. Secondary and tertiary structure of mutants were analyzed by CD and fluorescence spectroscopy. Our result illustrated that replacement of residues in the substrate-binding pocket would change the specificity and efficiency of variants.