Pharmacological p38 MAPK inhibitor SB203580 enhances AML stem cell line KG1a chemosensitivity to daunorubicin by promoting late apoptosis, cell growth arrest in S-phase, and miR-328-3p upregulation.

Acute myeloid leukaemia (AML) is characterized by uncontrolled proliferation of myeloid progenitor cells and impaired maturation, leading to immature cell accumulation in the bone marrow and bloodstream, resulting in hematopoietic dysfunction. Chemoresistance, hyperactivity of survival pathways, and miRNA alteration are major factors contributing to treatment failure and poor outcomes in AML patients. This study aimed to investigate the impact of the pharmacological p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 on the chemoresistance potential of AML stem cell line KG1a to the therapeutic drug daunorubicin (DNR). KG1a and chemosensitive leukemic HL60 cells were treated with increasing concentrations of DNR. Cell Titer-Glo®, flow cytometry, phosphokinase and protein arrays, Western blot technology, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were employed for assessment of cell viability, half-maximal inhibitory concentration (IC50) determination, apoptotic status detection, cell cycle analysis, apoptosis-related protein and gene expression monitoring. Confocal microscopy was used to visualize caspase and mitochondrial permeability transition pore (mPTP) activities. Exposed at various incubation times, higher DNR IC50 values were determined for KG1a cells than for HL60 cells, confirming KG1a cell chemoresistance potential. Exposed to DNR, late apoptosis induction in KG1a cells was enhanced after SB203580 pretreatment, defined as the combination treatment. This enhancement was confirmed by increased cleavage of poly(ADP-ribose) polymerase, caspase-9, caspase-3, and augmented caspase-3/-7 and mPTP activities in KG1a cells upon combination treatment, compared to DNR. Using phosphokinase and apoptosis protein arrays, the combination treatment decreased survival Akt phosphorylation and anti-apoptotic Bcl-2 expression levels in KG1a cells while increasing the expression levels of the tumor suppressor p53 and cyclin-dependent kinase inhibitor p21, compared to DNR. Cell cycle analysis revealed KG1a cell growth arrest in G2/M-phase caused by DNR, while combined treatment led to cell growth arrest in S-phase, mainly associated with cyclin B1 expression levels. Remarkably, the enhanced KG1a cell sensitivity to DNR after SB203580 pretreatment was associated with an increased upregulation of miR-328-3p and slight downregulation of miR-26b-5p, compared to DNR effect. Altogether, these findings could contribute to the development of a new therapeutic strategy by targeting the p38 MAPK pathway to improve treatment outcomes in patients with refractory or relapsed AML.

Blockade of p38 MAPK overcomes AML stem cell line KG1a resistance to 5-Fluorouridine and the impact on miRNA profiling.

Most of the AML patients in remission develop multidrug resistance after the first-line therapy and relapse. AML stem cells have gained attention for their chemoresistance potentials. Chemoresistance is a multifactorial process resulting from altered survival signaling pathways and apoptosis regulators such as MAPK, NF-κB activation and ROS production. We targeted the survival pathway p38 MAPK, NF-κB and ROS generation in human chemoresistant AML stem cell line KG1a, susceptible to enhance cell sensitivity to the chemotherapy drug 5-Fluorouridine, compared to the chemosensitive AML cell line HL60. After confirming the phenotypic characterization of KG1a and HL60 cells using flow cytometry and transcriptomic array analyses, cell treatment with the NF-κB inhibitor IKKVII resulted in a complete induction of apoptosis, and a few p38 MAPK inhibitor SB202190-treated cells underwent apoptosis. No change in the apoptosis status was observed in the ROS scavenger N-acetylcysteine-treated cells. The p38 MAPK pathway blockade enhanced the KG1a cell sensitivity to 5-Fluorouridine, which was associated with the upregulation of microribonucleic acid-(miR-)328-3p, as determined by the microarray-based miRNA transcriptomic analysis. The downregulation of the miR-210-5p in SB202190-treated KG1a cells exposed to FUrd was monitored using RT-qPCR. The miR-328-3p is known for the enhancement of cancer cell chemosensitivity and apoptosis induction, and the downregulation of miR-210-5p is found in AML patients in complete remission. In conclusion, we highlighted the key role of the p38 MAPK survival pathway in the chemoresistance capacity of the AML stem cells and potentially involved miRNAs, which may pave the way for the development of a new therapeutic strategy targeting survival signaling proteins and reduce the rate of AML relapse.

A Drug Repositioning Approach Identifies a Combination of Compounds as a Potential Regimen for Chronic Lymphocytic Leukemia Treatment.

Drug repositioning is a promising and powerful innovative strategy in the field of drug discovery. In this study, we screened a compound-library containing 800 Food and Drug Administration approved drugs for their anti-leukemic effect. All screening activities made use of human peripheral blood mononuclear cells (PBMCs), isolated from healthy or leukemic donors. Compounds with confirmed cytotoxicity were selected and classified in three groups: i) anti-neoplastic compounds which are drugs used in leukemia treatment, ii) compounds known to have an anti-cancer effect and iii) compounds demonstrating an anti-leukemic potential for the first time. The latter group was the most interesting from a drug repositioning perspective and yielded a single compound, namely Isoprenaline which is a non-selective ß-adrenergic agonist. Analysis of the cytotoxic effect of this drug indicated that it induces sustainable intracellular ATP depletion leading, over time, to necrotic cell death. We exploited the Isoprenaline-induced intracellular ATP depletion to sensitize primary leukemic cells to fludarabine (purine analogue) and Ibrutinib (Bruton's tyrosine kinase inhibitor) treatment. In-vitro treatment of primary leukemic cells with a combination of Isoprenaline/fludarabine or Isoprenaline/Ibrutinib showed a very high synergistic effect. These combinations could constitute a new efficient regimen for CLL treatment following successful evaluation in animal models and clinical trials.

Distinct anti-proliferative effects of herbal melanin on human acute monocytic leukemia THP-1 cells and embryonic kidney HEK293 cells.

BACKGROUND: Herbal melanin (HM) is a dark pigment extracted from the seed coat of Nigella sativa L. and known to exert biological effects via toll-like receptor 4 (TLR4). Recently, TLR4 was described as involved in natural programmed cell death (apoptosis). Tumor and embryonic cells are used as in vitro cellular models for drug and anti-cancer agent screening. To date, no cytotoxic studies have been reported of HM in TLR4-positive acute monocytic leukemia THP-1 cells compared to TLR4-negative human embryonic kidney HEK293 cells. METHODS: We studied the anti-proliferative effects of several HM concentrations on THP-1 and HEK293 cells by evaluating cell viability using the CellTiter-Glo® luminescent assay, assessing the TLR4 expression level, determining the apoptotic status, and analyzing the cell cycle distribution using flow cytometry. Apoptotic pathways were investigated using mitochondrial transition pore opening, caspase activity assays and immunoblot technology. RESULTS: Low HM concentrations did not affect THP-1 cell viability, but high HM concentrations (62.5-500 µg/mL) did decrease THP-1 cell viability and induced G0/G1 phase cell cycle arrest. Only at the highest concentration (500 µg/mL), HM slightly increased the TLR4 expression on the THP-1 cell surface, concomitantly upregulated TLR4 whole protein and gene expression, and induced apoptosis in THP-1 cells via activation of the extrinsic and intrinsic pathways. No change of apoptotic status was noticed in TLR4-negative HEK293 cells, although HM decreased HEK293 cell viability and induced cell growth arrest in the G2 phase. CONCLUSION: HM exerts distinct anti-proliferative effects on human acute monocytic leukemia and embryonic kidney cells mainly through cell cycle interference in a TLR4-independent manner and through apoptosis induction in a TLR4-dependent manner, as observed in only the THP-1 cells.

Preparation of iron oxide mesoporous magnetic microparticles as novel multidrug carriers for synergistic anticancer therapy and deep tumor penetration.

The preparation of mesoporous iron oxides with controllable physiochemical properties for effective therapeutic drug delivery remains a formidable challenge. Herein, iron oxide mesoporous magnetic microparticles (IO-MMMs) were prepared by a modified reverse hard-templating approach using, for the first time, acid-prepared mesoporous spheres (APMS) as the hard silica template. The obtained mesostructures exhibited remarkably high surface area and large pore volumes (SBET = 240 m2/g and Vpore = 0.55 cm3/g), controllable average sizes, generally uniform morphologies, and excellent biocompatibilities, allowing them to achieve optimal drug release in cancer cells and tumor tissues. IO-MMM carriers were able to co-load high amounts of hydrophilic chemotherapeutic drugs (Dox or Daun) and/or hydrophobic hormonal anticancer drugs (Tam), and release them sustainably in a pH-dependent manner, utilizing the fluorescence of Daun to real-time trace the intracellular drug distribution, and employing Daun/Tam to treat cancer by combined chemo/hormonal therapy. Cytotoxicity assays against different types of cancerous cells showed that the combinatory Daun/Tam@IO-MMM formulation significantly reduced the viability of metastatic MCF7 and KAIMRC1 breast as well as HCT8 colorectal cancer cells, with the least potency towards non-cancerous normal primary cells (up to 10-fold). Electron, flow, and live confocal microscopy imaging confirmed that the loaded vehicles were successfully and differentially uptaken by the different tested cells, gradually releasing their payloads, and causing apoptotic cell death. Importantly, compared to free drugs, Daun/Tam@IO-MMMs displayed enhanced drug accumulation in patient breast primary tumor tissues, deeply penetrating into the tumor region and killing the tumor cells inside. The designed carriers described here, thus, constitute a novel promising magnetic mesoporous smart system that entraps different kinds of drugs and release them in a controlled manner for combinatorial chemo/hormonal cancer theranostics. This multifactorial platform may open new avenues in cancer therapy as efficient synergistic antitumor system through overcoming limitations of conventional cancer therapy.

P19 Cells as a Model for Studying the Circadian Clock in Stem Cells before and after Cell Differentiation.

In mammals, circadian rhythmicity is sustained via a transcriptional/translational feedback loop referred to as the canonical molecular circadian clock. Circadian rhythm is absent in undifferentiated embryonic stem cells; it begins only after differentiation. We used pluripotent P19 embryonal carcinoma stem cells to check the biological clock before and after differentiation into neurons using retinoic acid. We show that the central clock genes ARNTL (Bmal), Per2 and Per3, and the peripheral clock genes Rev-erb-α and ROR-α, oscillate before and after differentiation, as does the expression of the neuronal differentiation markers Hes5, ß-3-tubulin (Tubb3) and Stra13, but not Neurod1. Furthermore, the known clock-modulating compounds ERK, EGFR, Pi3K, p38, DNA methylation and Sirtiun inhibitors, in addition to Rev-erb-α ligands, modulate the expression of central and peripheral clock genes. Interestingly Sirtinol, Sirt1 and Sirt2 inhibitors had the greatest significant effect on the expression of clock genes, and increased Hes5 and Tubb3 expression during neuronal differentiation. Our findings reveal a new frontier of circadian clock research in stem cells: contrary to what has been published previously, we have shown the clock to be functional and to oscillate, even in undifferentiated stem cells. Modulating the expression of clock genes using small molecules could affect stem cell differentiation.

Biological impact of advanced glycation endproducts on estrogen receptor-positive MCF-7 breast cancer cells.

Diabetes mellitus potentiates the risk of breast cancer. We have previously described the pro-tumorigenic effects of advanced glycation endproducts (AGEs) on estrogen receptor (ER)-negative MDA-MB-231 breast cancer cell line mediated through the receptor for AGEs (RAGE). However, a predominant association between women with ER-positive breast cancer and type 2 diabetes mellitus has been reported. Therefore, we have investigated the biological impact of AGEs on ER-positive human breast cancer cell line MCF-7 using in vitro cell-based assays including cell count, migration, and invasion assays. Western blot, FACS analyses and quantitative real time-PCR were also performed. We found that AGEs at 50-100µg/mL increased MCF-7 cell proliferation and cell migration associated with an enhancement of pro-matrix metalloproteinase (MMP)-9 activity, without affecting their poor invasiveness. However, 200µg/mL AGEs inhibited MCF-7 cell proliferation through induction of apoptosis indicated by caspase-3 cleavage detected using Western blotting. A phospho-protein array analysis revealed that AGEs mainly induce the phosphorylation of extracellular-signal regulated kinase (ERK)1/2 and cAMP response element binding protein-1 (CREB1), both signaling molecules considered as key regulators of AGEs pro-tumorigenic effects. We also showed that AGEs up-regulate RAGE and ER expression at the protein and transcript levels in MCF-7 cells, in a RAGE-dependent manner after blockade of AGEs/RAGE interaction using neutralizing anti-RAGE antibody. Throughout the study, BSA had no effect on cellular processes. These findings pave the way for future studies investigating whether the exposure of AGEs-treated ER-positive breast cancer cells to estrogen could lead to a potentiation of breast cancer development and progression.

Differential Expression of Circadian Genes in Leukemia and a Possible Role for Sirt1 in Restoring the Circadian Clock in Chronic Myeloid Leukemia.

Disregulation of genes making up the mammalian circadian clock has been associated with different forms of cancer. This study aimed to address how the circadian clock genes behave over the course of treatment for both the acute and chronic forms of leukemia and whether any could be used as potential biomarkers as a read-out for therapeutic efficacy. Expression profiling for both core and ancillary clock genes revealed that the majority of clock genes are down-regulated in acute myeloid leukemia patients, except for Cry2, which is up-regulated towards the end of treatment. A similar process was seen in acute lymphocytic leukemia patients; however, here, Cry2 expression came back up towards control levels upon treatment completion. In addition, all of the core clock genes were down-regulated in both chronic forms of leukemia (chronic myeloid leukemia and chronic lymphocytic leukemia), except for Cry2, which was not affected when the disease was diagnosed. Furthermore, the NAD(+) - dependent protein deacetylase Sirt1 has been proposed to have a dual role in both control of circadian clock circuitry and promotion of cell survival by inhibiting apoptotic pathways in cancer. We used a pharmacological-based approach to see whether Sirt1 played a role in regulating the circadian clock circuitry in both acute and chronic forms of leukemia. Our results suggest that interfering with Sirt1 leads to a partial restoration of BMAL1 oscillation in chronic myeloid leukemia patient samples. Furthermore, interfering with Sirt1 activity led to both the induction and repression of circadian clock genes in both acute and chronic forms of leukemia, which makes it a potential therapeutic target to either augment existing therapies for chronic leukemia or to act as a means of facilitating chronotherapy in order to maximize both the effectiveness of existing therapies and to minimize therapy-associated toxicity.