Characterisation of the cell and molecular biological effect of peptide-based daunorubicin conjugates developed for targeting pancreatic adenocarcinoma (PANC-1) cell line.

Pancreatic adenocarcinoma is one of the tumours with the worst prognosis, with a 5-year survival rate of 5-10%. Our aim was to find and optimise peptide-based drug conjugates with daunorubicin (Dau) as the cytotoxic antitumour agent. When conjugated with targeting peptides, the side effect profile and pharmacokinetics of Dau can be improved. The targeting peptide sequences (e.g. GSSEQLYL) we studied were originally selected by phage display. By Ala-scan technique, we identified that position 6 in the parental sequence (Dau=Aoa-LRRY-GSSEQLYL-NH2, ConjA) could be modified without the loss of antitumour activity (Dau=Aoa-LRRY-GSSEQAYL-NH2, Conj03: 14. 9% viability). Our results showed that the incorporation of p-chloro-phenylalanine (Dau=Aoa-LRRY-GSSEQF(pCl)YL-NH2, Conj16) further increased the antitumour potency (10-5 M: 9.7% viability) on pancreatic adenocarcinoma cells (PANC-1). We found that conjugates containing modified GSSEQLYL sequences could be internalised to PANC-1 cells and induce cellular senescence in the short term and subsequent apoptotic cell death. Furthermore, the cardiotoxic effect of Dau was markedly reduced in the form of peptide conjugates. In conclusion, Conj16 had the most effective antitumor activity on PANC-1 cells, which makes this conjugate promising for developing new targeted therapies without cardiotoxic effects.

Anthracyclines induce cardiotoxicity through a shared gene expression response signature.

TOP2 inhibitors (TOP2i) are effective drugs for breast cancer treatment. However, they can cause cardiotoxicity in some women. The most widely used TOP2i include anthracyclines (AC) Doxorubicin (DOX), Daunorubicin (DNR), Epirubicin (EPI), and the anthraquinone Mitoxantrone (MTX). It is unclear whether women would experience the same adverse effects from all drugs in this class, or if specific drugs would be preferable for certain individuals based on their cardiotoxicity risk profile. To investigate this, we studied the effects of treatment of DOX, DNR, EPI, MTX, and an unrelated monoclonal antibody Trastuzumab (TRZ) on iPSC-derived cardiomyocytes (iPSC-CMs) from six healthy females. All TOP2i induce cell death at concentrations observed in cancer patient serum, while TRZ does not. A sub-lethal dose of all TOP2i induces limited cellular stress but affects calcium handling, a function critical for cardiomyocyte contraction. TOP2i induce thousands of gene expression changes over time, giving rise to four distinct gene expression response signatures, denoted as TOP2i early-acute, early-sustained, and late response genes, and non-response genes. There is no drug- or AC-specific signature. TOP2i early response genes are enriched in chromatin regulators, which mediate AC sensitivity across breast cancer patients. However, there is increased transcriptional variability between individuals following AC treatments. To investigate potential genetic effects on response variability, we first identified a reported set of expression quantitative trait loci (eQTLs) uncovered following DOX treatment in iPSC-CMs. Indeed, DOX response eQTLs are enriched in genes that respond to all TOP2i. Next, we identified 38 genes in loci associated with AC toxicity by GWAS or TWAS. Two thirds of the genes that respond to at least one TOP2i, respond to all ACs with the same direction of effect. Our data demonstrate that TOP2i induce thousands of shared gene expression changes in cardiomyocytes, including genes near SNPs associated with inter-individual variation in response to DOX treatment and AC-induced cardiotoxicity.

The influence of cytotoxic drugs on the immunophenotype of blast cells in paediatric B precursor acute lymphoblastic leukaemia.

BACKGROUND: Flow cytometry plays is important in the diagnosis of acute lymphoblastic leukaemia (ALL) and when antigen-specific immunotherapy is indicated. We have investigated the effects of prednisolone, vincristine, daunorubicin, asparaginase and methotrexate on the antigen expression on blast cells that could influence the planning of antigen-specific therapy as well as risk-based treatment assignment. PATIENTS AND METHODS: Patients aged ≤ 17 years with de novo B-cell ALL (B-ALL) were enrolled in the study. Blast cells were isolated and exposed in vitro to 5 individual cytotoxic drugs in logarithmically increasing concentrations. Then, the expression of CD10, CD19, CD20, CD27, CD34, CD45, CD58, CD66c and CD137 antigens was determined by quantitative flow cytometry. RESULTS: Cytotoxic drugs caused dose-dependent or dose-independent modulation of antigen expression. Daunorubicin caused a dose-dependent down-modulation of CD10, CD19, CD34, CD45 and CD58 and an up-modulation of CD137. Vincristine caused a dose-dependent down-modulation of CD19 and CD58 and an up-modulation of CD45. Daunorubicin also caused dose-independent down-modulation of CD27 and prednisolone down-modulation of CD10, CD19, CD27, CD34 and CD58. Down-modulation of CD20 was detected only in relation to the specific dose of daunorubicin. CONCLUSIONS: The results of the study have shown that cytotoxic drugs can alter the expression of antigens that are important for immunotherapy. Importantly, daunorubicin, prednisolone and vincristine caused down-modulation of CD19 and CD58, suggesting that these drugs are better avoided during bridging therapy prior to bispecific antibodies or CAR-T cell therapy. In addition, immunophenotypic changes on blast cells induced by different drugs could also influence risk-based treatment assignment.

Natural products from Streptomyces spp. as potential inhibitors of the major factors (holoRdRp and nsp13) for SARS-CoV-2 replication: an in silico approach.

The COVID-19 pandemic caused unprecedented damage to humanity, and while vaccines have been developed, they are not fully effective against the SARS-CoV-2 virus. Limited targeted drugs, such as Remdesivir and Paxlovid, are available against the virus. Hence, there is an urgent need to explore and develop new drugs to combat COVID-19. This study focuses on exploring microbial natural products from soil-isolated bacteria Streptomyces sp. strain 196 and RI.24 as a potential source of new targeted drugs against SARS-CoV-2. Molecular docking studies were performed on holoRdRp and nsp13, two key factors responsible for virus replication factor. Our in silico studies, K-252-C aglycone indolocarbazole alkaloid (K252C) and daunorubicin were found to have better binding affinities than the respective control drugs, with K252C exhibiting binding energy of - 9.1 kcal/mol with holoRdRp and - 9.2 kcal/mol with nsp13, and daunorubicin showing binding energy at - 8.1 kcal/mol with holoRdRp and - 9.3 kcal/mol with nsp13. ADMET analysis, MD simulation, and MM/GBSA studies indicated that K252C and daunorubicin have the potential to be developed as targeted drugs against SARS-CoV-2. The study concludes that K252C and daunorubicin are potential lead compounds that might suppress the inhibition of SARS-CoV-2 replication among the tested microbial compounds and could be developed as targeted drugs against COVID-19. In the future, further in vitro studies are required to validate these findings.

[Effect of TRIM59 Expression Interference on Daunorubicin Chemosensitivity of Chronic Myeloid Leukemia K562 Cells and Its Mechanism].

OBJECTIVE: To investigate the effect of tripipartite motif 59 (TRIM59) expression interference on the chemosensitivity of daunorubicin (DNR) in chronic myeloid leukemia (CML) K562 cells and the related molecular mechanism. METHODS: The expressions of TRIM59 mRNA in bone marrow tissues of patients with CML and K562 cells were detected by RT-qPCR. Liposome-based transfection technology was used to transfect TRIM59-specific siRNA (si-TRIM59) into K562 cells which then were treated with DNR. The proliferation and apoptosis of cells were detected by CCK-8 assay and flow cytometry, respectively, and the expressions of apoptosis-related protein and Wnt/ß-catenin signaling pathway-related protein were detected by Western blot. RESULTS: Compared with the bone marrow tissue of CML patients at the time of initial treatment, the expression of TRIM59 mRNA in bone marrow tissue of CML patients at the time of chemotherapy resistance was significantly increased (P <0.05). Compared with control group, the cell proliferation inhibition rate and apoptosis rate in si-TRIM59 group and DNR group were significantly increased (P <0.05), the expression of Bax, Caspase3 and Cleaved-Caspase3 protein were significantly increased (P <0.05), while the expressions of Bcl-2, Wnt3α, GSK-3ß protein and the ratio of p-ß-catenin/ß-catenin were significantly decreased (P <0.05). Compared with si-TRIM59 group and DNR group, the proliferation inhibition rate and apoptosis rate of si-TRIM59+DNR group were significantly increased (P <0.05), the expression of Bax, Caspase3 and Cleaved-Caspase3 protein were significantly increased, while the expression of Bcl-2, Wnt3α, GSK-3ß protein and the ratio of p-ß-catenin/ß-catenin were significantly decreased (P <0.05). CONCLUSION: TRIM59 expression interference may enhance the chemosensitivity of K562 cells to DNR, and its mechanism may be related to the regulation of Wnt/ß-catenin signaling pathway.

[Study on the Effect and Mechanism of Tetrandrine on Bone Marrow Mesenchymal Stem Cell-Mediated Drug Resistance in Leukemia].

OBJECTIVE: To explore the role of bone marrow mesenchymal stem cells (BMSC)，an essential element of the bone marrow microenvironment, in multidrug resistance(MDR) of K562 cells, as well as the reversal effect of tetrandrine (TET) on BMSC-mediated MDR and its potential mechanism. METHODS: A mixed co-culture system and a transwell co-culture system for BMSC and K562 cells were established, and the cells were divided into different groups and treated with daunorubicin (DNR) alone or combined with TET and DNR. The CCK-8 assay was used to detect the proliferation of K562 cells in each group, and the cell inhibition rate was calculated. Cytometric bead array (CBA) was used to detect the expression levels of IFN, IL-2, IL-6 and IL-10 in the supernatant of different groups. RT-qPCR and Western blot were used to detected the expression of STAT3 at mRNA and protein levels, respectively. RESULTS: Compared with K562+DNR group, the inhibition rate of DNR on K562 cell proliferation in K562+BMSC+DNR group was significantly decreased (P < 0.05), while the levels of IL-6 in the culture supernatant and phosphorylated STAT3 in K562 cells were significantly increased (P < 0.05). Compared with K562+BMSC+DNR group, the inhibition rate of DNR on K562 cell proliferation in K562+BMSC+DNR+TET group was significantly increased (P < 0.05), while the level of IL-6 and phosphorylated STAT3 was significantly decreased (P < 0.05). CONCLUSION: BMSC can promote the drug resistance of leukemia cells, and TET may reverse the BMSC-mediated drug resistance via inhibiting IL-6/STAT3 signaling pathway.

CALCRL induces resistance to daunorubicin in acute myeloid leukemia cells through upregulation of XRCC5/TYK2/JAK1 pathway.

Chemotherapy is the main treatment option for acute myeloid leukemia (AML), but acquired resistance of leukemic cells to chemotherapeutic agents often leads to difficulties in AML treatment and disease relapse. High calcitonin receptor-like (CALCRL) expression is closely associated with poorer prognosis in AML patients. Therefore, this study was performed by performing CALCRL overexpression constructs in AML cell lines HL-60 and Molm-13 with low CALCRL expression. The results showed that overexpression of CALCRL in HL-60 and Molm-13 could confer resistance properties to AML cells and reduce the DNA damage and cell cycle G0/G1 phase blocking effects caused by daunorubicin (DNR) and others. Overexpression of CALCRL also reduced DNR-induced apoptosis. Mechanistically, the Cancer Clinical Research Database analyzed a significant positive correlation between XRCC5 and CALCRL in AML patients. Therefore, the combination of RT-PCR and Western blot studies further confirmed that the expression levels of XRCC5 and PDK1 genes and proteins were significantly upregulated after overexpression of CALCRL. In contrast, the phosphorylation levels of AKT/PKCε protein, a downstream pathway of XRCC5/PDK1, were significantly upregulated. In the response study, transfection of overexpressed CALCRL cells with XRCC5 siRNA significantly upregulated the drug sensitivity of AML to DNR. The expression levels of PDK1 protein and AKT/PKCε phosphorylated protein in the downstream pathway were inhibited considerably, and the expression of apoptosis-related proteins Bax and cleaved caspase-3 were upregulated. Animal experiments showed that the inhibitory effect of DNR on the growth of HL-60 cells and the number of bone marrow invasions were significantly reversed after overexpression of CALCRL in nude mice. However, infection of XCRR5 shRNA lentivirus in HL-60 cells with CALCRL overexpression attenuated the effect of CALCRL overexpression and upregulated the expression of apoptosis-related proteins induced by DNR. This study provides a preliminary explanation for the relationship between high CALCRL expression and poor prognosis of chemotherapy in AML patients. It offers a more experimental basis for DNR combined with molecular targets for precise treatment in subsequent studies.

A novel anticancer quinolone, (R)-WAC-224, has anti-leukemia activities against acute myeloid leukemia.

Approximately 60%-80% of patients who achieve complete remission eventually relapse after conventional chemotherapy and have poor prognoses despite the recent advances of novel anticancer agents. Continuing development of more effective novel treatments for acute myeloid leukemia (AML) is necessary. We developed (R)-WAC-224 (R-WAC), which is an anticancer quinolone, targeting topoisomerase II. This study evaluated the anti-leukemia potential of R-WAC or racemic WAC-224 (WAC) in vitro and in vivo. R-WAC significantly inhibited the human AML cell line proliferation (MV4-11, HL60, and KG1a), which was comparable to daunorubicin and cytarabine, not affected by P-glycoprotein overexpression. WAC did neither increase serum troponin-T nor decrease the crypt numbers in the small intestine, indicating WAC was less toxic than doxorubicin. R-WAC monotherapy demonstrated prolonged survival in the AML mice model and inhibited tumor growth in the MV4-11 xenograft mice model. Moreover, the combination of R-WAC and cytarabine demonstrated more active anti-leukemia effects than daunorubicin and cytarabine. Finally, R-WAC inhibited the colony-forming abilities using primary AML cells. These results indicate that R-WAC is a promising therapeutic agent for AML.

Comparative study on ABCB1-dependent efflux of anthracyclines and their metabolites: consequences for cancer resistance.

1. ABCB1 (P-glycoprotein, MDR1) is one of the most important transporter involved in cancer multi-drug resistance. It also plays a significant role in cancer resistance against anthracyclines, an anticancer group of drugs, including doxorubicin and daunorubicin. Several intracellular enzymes metabolise anthracyclines to carbonyl-reduced, hydroxy metabolites, which have impaired cytotoxic properties. However, metabolite efflux by ABCB1 transporter is not well characterised, while it may be the mechanism responsible for the metabolites' lack of activity.2. In this study recombinant ABCB1 ATPase transporter assay; anthracyclines accumulation assay in resistant cells overexpressing ABCB1; and molecular modelling were used to investigate anthracyclines: doxorubicin and daunorubicin and their carbonyl-reduced metabolites (doxorubicinol, daunorubicinol) susceptibility for ABCB1-dependent efflux.3. Based on the kinetics parameters of ATPase activity of ABCB1, it was found that daunorubicinol exerted an exceptionally high potential for being effluxed by the ABCB1 transporter. ABCB1 significantly affected the accumulation pattern of studied chemicals in resistant cancer cells. Doxorubicin and daunorubicinol accumulation were influenced by the activity of ABCB1 modulator - valspodar.4. Results indicate that ABCB1 activity affects not only anthracyclines but also their metabolites. Therefore crosstalk between the process of anthracyclines metabolism and metabolite efflux may be the mechanism of impairing anticancer properties of anthracyclines metabolites.

Alternating electric fields can improve chemotherapy treatment efficacy in blood cancer cell U937 (non-adherent cells).

BACKGROUND: Recent achievements in cancer therapy are the use of alternating electrical fields at intermediate frequencies (100-300 kHz) and low intensities (1-3 V/cm), which specifically target cell proliferation while affecting different cellular activities depending on the frequency used. METHODS: In this article, we examine the effect of electric fields on spherical suspended cells and propose the combination of Daunorubicin, a chemotherapy agent widely used in the treatment of acute myeloid leukemia, with electric field exposure. U937 cells were subjected to an electric field with a frequency of 200 kHz and an intensity of 0.75 V/cm, or to a combination of Daunorubicin and electric field exposure, resulting in a significant reduction in cell proliferation. Furthermore, the application of an electric field to U937 cells increased Daunorubicin uptake. RESULTS: Apoptosis and DNA damage were induced by the electric field or in conjunction with Daunorubicin. Notably, normal cells exposed to an electric field did not show significant damage, indicating a selective effect on dividing cancer cells (U937). Moreover, the electric field affects the U937 cell line either alone or in combination with Daunorubicin. This effect may be due to increased membrane permeability. CONCLUSIONS: Our findings suggest that the use of electric fields at intermediate frequencies and low intensities, either alone or in combination with Daunorubicin, has potential as a selective anti-cancer therapy for dividing cancer cells, particularly in the treatment of acute myeloid leukemia. Further research is needed to fully understand the underlying mechanisms and to optimize the use of this therapy.

Transcriptional Response to Standard AML Drugs Identifies Synergistic Combinations.

Unlike genomic alterations, gene expression profiles have not been widely used to refine cancer therapies. We analyzed transcriptional changes in acute myeloid leukemia (AML) cell lines in response to standard first-line AML drugs cytarabine and daunorubicin by means of RNA sequencing. Those changes were highly cell- and treatment-specific. By comparing the changes unique to treatment-sensitive and treatment-resistant AML cells, we enriched for treatment-relevant genes. Those genes were associated with drug response-specific pathways, including calcium ion-dependent exocytosis and chromatin remodeling. Pharmacological mimicking of those changes using EGFR and MEK inhibitors enhanced the response to daunorubicin with minimum standalone cytotoxicity. The synergistic response was observed even in the cell lines beyond those used for the discovery, including a primary AML sample. Additionally, publicly available cytotoxicity data confirmed the synergistic effect of EGFR inhibitors in combination with daunorubicin in all 60 investigated cancer cell lines. In conclusion, we demonstrate the utility of treatment-evoked gene expression changes to formulate rational drug combinations. This approach could improve the standard AML therapy, especially in older patients.

DeSUMOylation of chromatin-bound proteins limits the rapid transcriptional reprogramming induced by daunorubicin in acute myeloid leukemias.

Genotoxicants have been used for decades as front-line therapies against cancer on the basis of their DNA-damaging actions. However, some of their non-DNA-damaging effects are also instrumental for killing dividing cells. We report here that the anthracycline Daunorubicin (DNR), one of the main drugs used to treat Acute Myeloid Leukemia (AML), induces rapid (3 h) and broad transcriptional changes in AML cells. The regulated genes are particularly enriched in genes controlling cell proliferation and death, as well as inflammation and immunity. These transcriptional changes are preceded by DNR-dependent deSUMOylation of chromatin proteins, in particular at active promoters and enhancers. Surprisingly, inhibition of SUMOylation with ML-792 (SUMO E1 inhibitor), dampens DNR-induced transcriptional reprogramming. Quantitative proteomics shows that the proteins deSUMOylated in response to DNR are mostly transcription factors, transcriptional co-regulators and chromatin organizers. Among them, the CCCTC-binding factor CTCF is highly enriched at SUMO-binding sites found in cis-regulatory regions. This is notably the case at the promoter of the DNR-induced NFKB2 gene. DNR leads to a reconfiguration of chromatin loops engaging CTCF- and SUMO-bound NFKB2 promoter with a distal cis-regulatory region and inhibition of SUMOylation with ML-792 prevents these changes.

Effect of siRNA-mediated silencing of p53R2 gene on sensitivity of T-ALL cellsto Daunorubicin.

INTRODUCTION: p53R2 is a p53-inducible protein that, as one of the subunits of ribonucleotide reductase, plays an important role in providing dNTPs for DNA repair. Although p53R2 is associated with cancer progression, its role in T-cell acute lymphoblastic leukemia (T-ALL) cells is unknown. Therefore, in this study, we evaluated the effect of p53R2 silencing on double-stranded DNA breaks, apoptosis and cell cycle of T-ALL cells treated with Daunorubicin. METHODS: Transfection was performed using Polyethyleneimine (PEI). Gene expression was measured using real-time PCR and protein expression was evaluated using Western blotting. Cell metabolic activity and IC50 were calculated using MTT assay, formation of double-stranded DNA breaks was checked using immunohistochemistry for γH2AX, and cell cycle and apoptosis were evaluated using flow cytometry. RESULTS: We found that p53 silencing synergistically inhibited the growth of T-ALL cells by Daunorubicin. p53R2 siRNA in combination with Daunorubicin but not alone increases the rate of DNA double-strand breaks in T-ALL cells. In addition, p53R2 siRNA significantly increased Daunorubicin-induced apoptosis. p53R2 siRNA also caused a non-significant increase in cells in G2 phase. CONCLUSION: The results of the present study showed that silencing of p53R2 using siRNA can significantly increase the antitumor effects of Daunorubicin on T-ALL cells. Therefore, p53R2 siRNA has the potential to be used as an adjuvant therapy in combination with Daunorubicin in T-ALL.

Chloroquine enhances the efficacy of chemotherapy drugs against acute myeloid leukemia by inactivating the autophagy pathway.

Current therapy for acute myeloid leukemia (AML) is largely hindered by the development of drug resistance of commonly used chemotherapy drugs, including cytarabine, daunorubicin, and idarubicin. In this study, we investigated the molecular mechanisms underlying the chemotherapy drug resistance and potential strategy to improve the efficacy of these drugs against AML. By analyzing data from ex vivo drug-response and multi-omics profiling public data for AML, we identified autophagy activation as a potential target in chemotherapy-resistant patients. In THP-1 and MV-4-11 cell lines, knockdown of autophagy-regulated genes ATG5 or MAP1LC3B significantly enhanced AML cell sensitivity to the chemotherapy drugs cytarabine, daunorubicin, and idarubicin. In silico screening, we found that chloroquine phosphate mimicked autophagy inactivation. We showed that chloroquine phosphate dose-dependently down-regulated the autophagy pathway in MV-4-11 cells. Furthermore, chloroquine phosphate exerted a synergistic antitumor effect with the chemotherapy drugs in vitro and in vivo. These results highlight autophagy activation as a drug resistance mechanism and the combination therapy of chloroquine phosphate and chemotherapy drugs can enhance anti-AML efficacy.

In Silico and In Vitro Assessment of Carbonyl Reductase 1 Inhibition Using ASP9521-A Potent Aldo-Keto Reductase 1C3 Inhibitor with the Potential to Support Anticancer Therapy Using Anthracycline Antibiotics.

Anthracycline antibiotics (ANT) are among the most widely used anticancer drugs. Unfortunately, their use is limited due to the development of drug resistance and cardiotoxicity. ANT metabolism, performed mainly by two enzymes-aldo-keto reductase 1C3 (AKR1C3) and carbonyl reductase 1 (CBR1)-is one of the proposed mechanisms generated by the described effects. In this study, we evaluated the CBR1 inhibitory properties of ASP9521, a compound already known as potent AKR1C3 inhibitor. First, we assessed the possibility of ASP9521 binding to the CBR1 catalytic site using molecular docking and molecular dynamics. The research revealed a potential binding mode of ASP9521. Moderate inhibitory activity against CBR1 was observed in studies with recombinant enzymes. Finally, we examined whether ASP9521 can improve the cytotoxic activity of daunorubicin against human lung carcinoma cell line A549 and assessed the cardioprotective properties of ASP9521 in a rat cardiomyocytes model (H9c2) against doxorubicin- and daunorubicin-induced toxicity. The addition of ASP9521 ameliorated the cytotoxic activity of daunorubicin and protected rat cardiomyocytes from the cytotoxic effect of both applied drugs. Considering the favorable bioavailability and safety profile of ASP9521, the obtained results encourage further research. Inhibition of both AKR1C3 and CBR1 may be a promising method of overcoming ANT resistance and cardiotoxicity.

Pinostrobin induces acute leukemia cell apoptosis via the regulation of miR-410-5p and SFRP5.

AIMS: This study attempted to explore the mechanisms involved in pinostrobin (PN)-mediated acute leukemia cell apoptosis regulated by miR-410-5p. MATERIAL AND METHODS: NB4 and MOLT-4 cells were cultured and treated with PN at the IC50 concentration. Apoptosis was examined by Annexin V-FITC/PI staining. RT-qPCR was used to measure the expression of caspase-3, BAK, BCL-W, and MCL-1. The target protein of PN was identified using LC-MS/MS followed by bioinformatic analysis. TargetScan, DIANA, and miRDB were used for the prediction of miRNAs involved in the PN-induced apoptosis mechanism. miRNA mimic transfection, RT-qPCR, and western blot analysis were performed to evaluate the regulatory effect of miRNA on its target and the involvement of miRNA in apoptosis induction by PN. In addition, the synergistic effect of PN and daunorubicin (DNR) were investigated by using the MTT assay. KEY FINDINGS: The results showed that PN reduced cell viability and induced apoptosis in both leukemia cell lines. From the LC-MS/MS and bioinformatics analysis, SFRP5 and miR-410-5p were selected as a potential PN target protein and miRNA, respectively. After miRNA mimic transfection, miR-410-5p, which is an onco-miRNA, was decreased and led to increased apoptosis in both cell lines, indicating that this miRNA is involved in PN-mediated apoptosis mechanisms. Moreover, PN demonstrated a synergistic effect with DNR, suggesting that PN may be used in combination with conventional chemotherapy drugs. SIGNIFICANCE: PN regulates the expression of miR-410-5p and SFRP5 to promote apoptosis in acute leukemia cells. It could be developed as an alternative treatment for leukemia in the future.

The Anticancer Drug Daunomycin Directly Affects Gene Expression and DNA Structure.

Daunomycin (DM), an anthracycline antibiotic, is frequently used to treat various cancers, but the direct effects of DM on gene expression and DNA structure are unclear. We used an in vitro cell-free system, optimized with spermine (SP), to study the effect of DM on gene expression. A bimodal effect of DM on gene expression, weak promotion followed by inhibition, was observed with increasing concentration of DM. We also performed atomic force microscopy observation to measure how DM affects the higher-order structure of DNA induced with SP. DM destroyed SP-induced flower-like conformations of DNA by generating double-strand breaks, and this destructive conformational change of DNA corresponded to the inhibitory effect on gene expression. Interestingly, the weakly enhanced cell-free gene expression occurred as DNA conformations were elongated or relaxed at lower DM concentrations. We expect these newly unveiled DM effects on gene expression and the higher-order structure of DNA will contribute further to the development and refinement of useful anticancer therapy chemicals.

Spectroscopic, calorimetric and cytotoxicity studies on the combined binding of daunorubicin and acridine orange to a DNA tetrahedron.

Chemotherapy-phototherapy (CTPT) combination drugs co-loaded by targeted DNA nanostructures can achieve controlled drug delivery, reduce toxic side effects and overcome multidrug resistance. Herein, we constructed and characterized a DNA tetrahedral nanostructure (MUC1-TD) linked with the targeting aptamer MUC1. The interaction of daunorubicin (DAU)/acridine orange (AO) alone and in combination with MUC1-TD and the influence of the interaction on the cytotoxicity of the drugs were evaluated. Potassium ferrocyanide quenching analysis and DNA melting temperature assays were used to demonstrate the intercalative binding of DAU/AO to MUC1-TD. The interactions of DAU and/or AO with MUC1-TD were analyzed by fluorescence spectroscopy and differential scanning calorimetry. The number of binding sites, binding constant, entropy and enthalpy changes of the binding process were obtained. The binding strength and binding sites of DAU were higher than those of AO. The presence of AO in the ternary system weakened the binding of DAU to MUC1-TD. In vitro cytotoxicity studies demonstrated that the loading of MUC1-TD augmented the inhibitory effects of DAU and AO and the synergistic cytotoxic effects of DAU + AO on MCF-7 cells and MCF-7/ADR cells. Cell uptake studies showed that the loading of MUC1-TD was beneficial in promoting the apoptosis of MCF-7/ADR cells due to its enhanced targeting to the nucleus. This study has important guiding significance for the combined application of DAU and AO co-loaded by DNA nanostructures to overcome multidrug resistance.

A comparative analysis of daunorubicin and its metabolite daunorubicinol interaction with apoptotic and drug resistance proteins using in silico approach.

Daunorubicin (DNR) is a chemotherapeutic drug associated with multiple side effects, including drug resistance. As the molecular mechanism related to these side effects remain unclear and mostly hypothesized, this study addresses and compares the role of DNR and its metabolite Daunorubicinol (DAUNol) to induce apoptosis and drug resistance using molecular docking, Molecular Dynamics (MD) simulation, MM-PBSA and chemical pathway analysis. The results showed that DNR's interaction was stronger with Bax protein, Mcl-1:mNoxaB and Mcl-1:Bim protein complexes than DAUNol. On the other hand, contrasting results were obtained for drug resistance proteins where stronger interaction was obtained with DAUNol compared to DNR. Further, MD simulation performed for 100 ns provided the details of protein-ligand interaction. Most notable was the interaction of Bax protein with DNR, resulting in conformational changes at α-helices 5, 6 and 9, leading to Bax activation. Finally, the chemical signalling pathway analysis also revealed the regulation of different signalling pathways by DNR and DAUNol. It was observed that DNR majorly impacted the signalling associated with apoptosis while DAUNol mainly targeted pathways related to multidrug resistance and cardiotoxicity. Overall, the results highlight that DNR biotransformation reduces its capability to induce apoptosis while enhancing its ability to induce drug resistance and off-target toxicity.Communicated by Ramaswamy H. Sarma.

DNMT3A low-expression is correlated to poor prognosis in childhood B-ALL and confers resistance to daunorubicin on leukemic cells.

BACKGROUND: Little is known about DNMT3A expression and its prognostic significance in childhood B cell acute lymphoblastic leukemia (B-ALL). METHODS: We determined DNMT3A mRNA expression in 102 children with B-ALL. Correlations with relapse-free survival (RFS) and common clinical characteristics were analyzed. DNMT3A was stably knocked out by CRISPR/Cas9 gene editing technology in Reh and 697 B-ALL cell lines. Cell proliferation activity after treated with daunorubicin (DNR) was determined by CCK8 assay in DNMT3A KO Reh and 697 cell lines. RESULTS: DNMT3A expression in B-ALL patients who were in continuous complete remission (CCR) was higher than in those who got relapse (P = 0.0111). Receiver operating characteristic curve showed prognostic significance of DNMT3A expression (P = 0.003). Low expression of DNMT3A (≤ 0.197) was significantly correlated with poor RFS (P < 0.001) in children with B-ALL. Knock-out of DNMT3A in Reh and 697 cell lines significantly increased IC50 of DNR (P = 0.0201 and 0.0022 respectively), indicating elevated resistance to DNR. CONCLUSION: Low expression of DNMT3A associates with poor prognosis in children with B-ALL. Knock-out of DNMT3A confers resistance to DNR on leukemic cells.