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Sweet syndrome (SS), also referred as acute febrile neutrophilic dermatosis, is an inflammatory process characterised by the abrupt appearance of erythematous papules or nodules with predominant neutrophilic infiltration in the dermis. Fever and neutrophilia are common presenting features. However, extracellular manifestations, including ocular and musculoskeletal, may occur. SS is divided into three subtypes: classical (or idiopathic), malignancy associated and drug induced. Medication-induced subtype accounts for up to 26% of cases. In recent years, emerging evidence has showed that SS may also occur in neutropenic patients who underwent induction for acute myeloid leukemia (AML). The identification of FMS-like tyrosine kinase 3 (FLT3) gene mutation in approximately 30% of patients with AML has promoted the targeted therapy with FLT3-internal tandem duplication (ITD) inhibitors. Midostaurin, a recently Food and Drug Administration-approved medication for FLT3-ITD-positive AML, was reported once as cause for SS. We report a midostaurin-induced SS with neutropenia in a patient following induction chemotherapy of AML

Background: 2-Furanones have attracted great attention due to their biological activities. They also have the ability to be converted to several biologically active heterocyclic and nonheterocyclic compounds, especially as anti-cancer agents. Objectives: This research aims to share in the development process of novel cytotoxic agents by synthesizing certain 2-furanone derivatives and using them as starting materials for the preparation of novel heterocyclic and non-heterocyclic compounds, then testing the synthesized derivatives for their anti-cancer activities. Methods: All the newly synthesized compounds were fully characterized by elemental analysis, IR, Mass, and 1H-NMR spectroscopy. 18 synthesized compounds were selected by National Cancer Institute (NCI) for testing against 60 cell lines, and the active compound was tested as MAPK14 and VEGFR2-inhibitor using Staurosporine as standard. Results: Compound 3a showed the higher activity against several cell lines; Leukemia (SR), Non- Small Cell Lung Cancer (NCI-H460), colon cancer (HCT-116), ovarian cancer (OVCAR-4), renal cancer (786-0, ACHN and UO-31) and, finally breast cancer (T-47D). It also has better inhibition activity against MAPK14 than the used reference. Conclusion: Compound 3a has promising anti-cancer activities compared to the used standards and may need further modification and investigations.

The aim of the work is to generalize the literature data on indolocarbazole derivatives with an antitumor activity.Materials and methods. The objects of the study were the preparations based on indolocarbazole derivatives with the antitumor activity. To search for materials on the problem under study, the following search and information as well as library databases were used: ebibrary, PubMed, CyberLeninka, ResearchGate, the State Register of Medicines, clinical trials registries clinline.ru and clinicaltrials.gov. The search for the following words / phrases was performed: indolocarbazoles, indolocarbazole derivatives, staurosporine, rebeccamycin, staurosporine derivatives. The search was conducted from January 11 until March 1, 2021. The compounds with a biological activity which were undergoing or had undergone preclinical and clinical trials, were taken into account. All the materials from 1977 to January 1, 2021, were taken into account.Results. The materials obtained indicate that indolocarbazole derivatives are promising compounds for the creation of anticancer medicinal preparations due to their properties and peculiarities of the action mechanism. These drugs have a selective action due to the targeted interaction with specific molecular targets: kinases (especially protein kinase C and its isozymes), DNA and DNA topoisomerase. To date, many compounds from the class of indolocarbazoles have been synthesized and investigated. They have shown a high antitumor activity in the treatment of systemic and solid tumors. However, despite this, only one MP based on a staurosporine derivative, registered by the TN of Rydapt® (in the USA and EU countries) and Miticaid® (in the Russian Federation), is approved for use in the clinical practice.Conclusion. Thus, the basic data from scientific publications on promising anticancer medicinal preparations based on compounds from the class of indolocarbazoles, have been summarized. The information is provided, in particular, on their molecular structure, the origin, classification, the main representatives of the class, which are at various stages of the research and are approved for use in the clinic.

Summary Introduction: There are many scientific reports on the beneficial effects of epigallocatechin gallate and resveratrol on the human body, e.g. antioxidant properties, a protective effect on the circulatory system and reduction of inflammation. Objective: The aim of the study was to evaluate the effect of these substances on the survival of mesenchymal stem cells (MSC) in the presence of the pro-apoptotic factor staurosporine. Methods: Cell viability WST-1 colorimetric assay. Results: It was confirmed that both 25 µM/ml and 50 µM/ml of epigallocatechin and 50 µM/ml of resveratrol statistically significantly increased the MSC survival rate. Conclusion: An excess supply of epigallocatechin gallate (50 µM/ml and higher) has a cytotoxic effect on MSC, which may have a negative impact on the body’s auto-regenerative capacity. Under toxic and stressful conditions, resveratrol and epigallocatechin gallate perform cytoprotective functions, thereby reducing the negative impact of toxic environmental conditions on the mesenchymal stem cells.

Axonal degeneration of retinal ganglion cells (RGCs) causes blindness in glaucoma. Currently, there are no therapies that target axons to prevent them from degenerating. Activation of the BAX protein has been shown to be the determining step in the intrinsic apoptotic pathway that causes RGCs to die in glaucoma. A putative role for BAX in axonal degeneration is less well elucidated. BCLXL (BCL2L1) is the primary antagonist of BAX in RGCs. We developed a mCherry-BCLXL fusion protein, which prevented BAX recruitment and activation to the mitochondria in tissue culture cells exposed to staurosporine. This fusion protein was then packaged into adeno-associated virus serotype 2, which was used to transduce RGCs after intravitreal injection and force its overexpression. Transduced RGCs express mCherry-BCLXL throughout their somas and axons along the entire optic tract. In a model of acute optic nerve crush, the transgene prevented the recruitment of a GFP-BAX fusion protein to mitochondria and provided long-term somal protection up to 12 weeks post injury. To test the efficacy in glaucoma, DBA/2J mice were transduced at 5 months of age, just prior to the time they begin to exhibit ocular hypertension. Gene therapy with mCherry-BCLXL did not affect the longitudinal history of intraocular pressure elevation compared to naive mice but did robustly attenuate both RGC soma pathology and axonal degeneration in the optic nerve at both 10.5 and 12 months of age. BCLXL gene therapy is a promising candidate for glaucoma therapy.

INTRODUCTION: Protein kinase C (PKC) is a promising drug target for various therapeutic areas. Natural products derived from plants, animals, microorganisms, and marine organisms have been used by humans as medicine from prehistoric times. Recently, several compounds derived from plants have been found to modulate PKC activities through competitive binding with ATP binding site, and other allosteric regions of PKC. As a result fresh race has been started in academia and pharmaceutical companies to develop an effective naturally derived small-molecule inhibitor to target PKC activities. Herein, in this review, we have discussed several natural products and their derivatives, which are reported to have an impact on PKC signaling cascade. METHODS: All information presented in this review article regarding the regulation of PKC by natural products has been acquired by a systematic search of various electronic databases, including ScienceDirect, Scopus, Google Scholar, Web of science, ResearchGate, and PubMed. The keywords PKC, natural products, curcumin, rottlerin, quercetin, ellagic acid, epigallocatechin-3 gallate, ingenol 3 angelate, resveratrol, protocatechuic acid, tannic acid, PKC modulators from marine organism, bryostatin, staurosporine, midostaurin, sangivamycin, and other relevant key words were explored. RESULTS: The natural products and their derivatives including curcumin, rottlerin, quercetin, ellagic acid, epigallocatechin-3 gallate, ingenol 3 angelate, resveratrol, bryostatin, staurosporine, and midostaurin play a major role in the management of PKC activity during various disease progression. CONCLUSION: Based on the comprehensive literature survey, it could be concluded that various natural products can regulate PKC activity during disease progression. However, extensive research is needed to circumvent the challenge of isoform specific regulation of PKC by natural products.

The carbohydrate response element‐binding protein (ChREBP), a glucose‐responsive transcription factor that plays a critical role in the glucose‐mediated induction of genes involved in hepatic glycolysis and lipogenesis, exists as two isoforms: ChREBPα and ChREBPβ. However, the mechanism responsible for regulating the expression of both ChREBPα and β, as well as the mechanism that determines which specific isoform is more responsive to different stimuli, remains unclear. To address this issue, we compared the effects of several stimuli, including oxidative stress, on the mRNA and protein expression levels of ChREBPα and β in the hepatocyte cell line, HepG2. We found that H2O2 stimulation suppressed the expression of both mRNA and protein in HepG2 cells, but the mRNA expression level of ChREBPβ was, The carbohydrate response element binding protein (ChREBP) is a glucose‐responsive transcription factor that strongly regulates glycolysis and lipogenesis. In this study, we showed that oxidative stress, including STS and TPA treatment, suppressed the expression of both ChREBPα and β through activation of ERK signaling in HepG2 cells.

Bastian Welz,* Rolf Bikker,* Leonie Hoffmeister, Mareike Diekmann, Martin Christmann, Korbinian Brand,* René Huber* Institute of Clinical Chemistry, Hannover Medical School, Hannover, 30625, Germany*These authors contributed equally to this workCorrespondence: Korbinian BrandInstitute of Clinical Chemistry, Hannover Medical School, Carl-Neuberg Str. 1, Hannover, 30625, GermanyTel +49 511 532 6614Fax +49 511 532 8614Email brand.korbinian@mh-hannover.deBackground: Termination of TNF-induced signaling plays a key role in the resolution of inflammation with dysregulations leading to severe pathophysiological conditions (sepsis, chronic inflammatory disease, cancer). Since a recent phospho-proteome analysis in human monocytes suggested GSK3 as a relevant kinase during signal termination, we aimed at further elucidating its role in this context.Materials and Methods: For the analyses, THP-1 monocytic cells and primary human monocytes were used. Staurosporine (Stauro) was applied to activate GSK3 by inhibiting kinases that mediate inhibitory GSK3α/β-Ser21/9 phosphorylation (eg, PKC). For GSK3 inhibition, Kenpaulone (Ken) was used. GSK3- and PKC-siRNAs were applied for knockdown experiments. Protein expression and phosphorylation were assessed by Western blot or ELISA and mRNA expression by qPCR. NF-κB activation was addressed using reporter gene assays.Results: Constitutive GSK3β and PKCβ expression and GSK3α/β-Ser21/9 and PKCα/βII-Thr638/641 phosphorylation were not altered during TNF long-term incubation. Stauro-induced GSK3 activation (demonstrated by Bcl3 reduction) prevented termination of TNF-induced signaling as reflected by strongly elevated IL-8 expression (used as an indicator) following TNF long-term incubation. A similar increase was observed in TNF short-term-exposed cells, and this effect was inhibited by Ken. PKCα/β-knockdown modestly increased, whereas GSK3α/β-knockdown inhibited TNF-induced IL-8 expression. TNF-dependent activation of two NF-κB-dependent indicator plasmids was enhanced by Stauro, demonstrating transcriptional effects. A TNF-induced increase in p65-Ser536 phosphorylation was further enhanced by Stauro, whereas IκBα proteolysis and IKKα/β-Ser176/180 phosphorylation were not affected. Moreover, PKCβ-knockdown reduced levels of Bcl3. A20 and IκBα mRNA, both coding for signaling inhibitors, were dramatically less affected under our conditions when compared to IL-8, suggesting differential transcriptional effects.Conclusion: Our results suggest that GSK3 activation is involved in preventing the termination of TNF-induced signaling. Our data demonstrate that activation of GSK3 – either pathophysiologically or pharmacologically induced – may destroy the finely balanced condition necessary for the termination of inflammation-associated signaling.Keywords: TNF, GSK3, PKC, staurosporine, IL-8, NF-κB, termination of TNF-induced signaling, termination of inflammation

Rapid identification of inhibitors for a family of proteins and prediction of ligand specificity are highly desirable for structure-based drug design. However, sequentially docking ligands into each protein target with conventional single-target docking methods is too computationally expensive to achieve these two goals, especially when the number of the targets is large. In this work, we use an efficient ensemble docking algorithm for simultaneous docking of ligands against multiple protein targets. We use protein kinases, a family of proteins that are highly important for many cellular processes and for rational drug design, as an example to demonstrate the feasibility of investigating ligand selectivity with this algorithm. Specifically, 14 human protein kinases were selected. First, native docking calculations were performed to test the ability of our energy scoring function to reproduce the experimentally determined structures of the ligand-protein kinase complexes. Next, cross-docking calculations were conducted using our ensemble docking algorithm to study ligand selectivity, based on the assumption that the native target of an inhibitor should have a more negative (i.e., favorable) energy score than the non-native targets. Staurosporine and Gleevec were studied as examples of nonselective and selective binding, respectively. Virtual ligand screening was also performed against five protein kinases that have at least seven known inhibitors. Our quantitative analysis of the results showed that the ensemble algorithm can be effective on screening for inhibitors and investigating their selectivities for multiple target proteins.

Contains fulltext : 237748.pdf (Publisher’s version ) (Closed access) The prospective randomized, placebo-controlled CALGB 10603/RATIFY trial (Alliance) demonstrated a statistically significant overall survival benefit from the addition of midostaurin to standard frontline chemotherapy in a genotypically-defined subgroup of 717 patients with FLT3-mutant acute myeloid leukemia (AML). The risk of death was reduced by 22% on the midostaurin-containing arm. In this post hoc analysis, we analyzed the cumulative incidence of relapse (CIR) on this study and also evaluated the impact of 12 4-week cycles of maintenance therapy. CIR analyses treated relapses and AML deaths as events, deaths from other causes as competing risks, and survivors in remission were censored. CIR was improved on the midostaurin arm (HR = 0.71 (95% CI, 0.54-0.93); p = 0.01), both overall and within European LeukemiaNet 2017 risk classification subsets when post-transplant events were considered in the analysis as events. However, when transplantation was considered as a competing risk, there was overall no significant difference between the risks of relapse on the two randomized arms. Patients still in remission after consolidation with high-dose cytarabine entered the maintenance phase, continuing with either midostaurin or placebo. Analyses were inconclusive in quantifying the impact of the maintenance phase on the overall outcome. In summary, midostaurin reduces the CIR.

This work synthesized the ceria oxide nanoparticles (CeO2 NPs) using the plant extract of Stachys japonica Miq and determined their antioxidant and antidiabetic activities. The characterization results demonstrated the successful formation of spherical shaped CeO2 NPs with average size of 21 nm with PDI of 0.121. The FTIR peak of CeO2 NPs revealed the capping of phyto-molecules and a peak at 470.92 cm−1 indicated the formation of Ce–O of CeO2 NPs. The CeO2 NPs scavenged the free radicals of ABTS+ and DPPH, with IC50 of 12.16 ± 0.12 μg. mL−1 and 109.5 ± 0.26 μg. mL−1 respectively. The 50 μg mL−1 of CeO2 NPs showed the maximum inhibitory effect on α-glucosidase (19.78 ± 0.95%) and α-amylase (43.24 ± 1.14%). The cellular assay revealed the non-toxicity of CeO2 NPs in NIH3T3 and HepG2 cells. Cell antioxidant assay demonstrated that CeO2 NPs attenuated the staurosporine (STS) induced-oxidative stress in NIH3T3 cells through preventing the MMP loss and nucleus damage. The treatment of CeO2 NPs was broken the insulin resistance in IR-HepG2 cells and increased the glucose uptake through strengthening the mitochondria from oxidative stress. These antioxidant and antidiabetic properties of CeO2 NPs have opened a new avenue for extending future study on CeO2 NPs regulated glucose metabolism and its underlying molecular mechanism for beneficial therapy of diabetes.

Tumor hypoxia induces the large-scale adaptive reprogramming of cancer cells, promoting their transformation into highly invasive and metastatic species that lead to highly negative prognoses for cancer patients. We describe the synthesis and application of a hypoxia-responsive trigger derived from previously inaccessible enamine N-oxide structures. Hypoxia-dependent reduction of this motif by hemeproteins results in the concomitant activation of a caged molecule and a latent electrophile. We exploit the former in a hypoxia-activated prodrug application using a caged staurosporine molecule as a proof-of-principle. We demonstrate the latter in in vivo tumor labeling applications with enamine-N-oxide-modified near-infrared probes. Hypoxia-activated prodrug development has long been complicated by the heterogeneity of tumor hypoxia in patients. The dual drug release and imaging modalities of the highly versatile enamine N-oxide motif present an attractive opportunity for theranostic development that can address the need not only for new therapeutics but paired methods for patient stratification.

Poly(ADP-ribose) polymerases-1 (PARP-1) are involved in DNA repair damage and so PARP-1 inhibitors have been used as potentiators in combination with DNA damaging cytotoxic agents to compromise the cancer cell DNA repair mechanism, resulting in genomic dysfunction and cell death. In this study, we report the synthesis of a novel series of pyrano[2,3-d]pyrimidine-2,4-dione analogues as potential inhibitors against PARP-1. All the newly synthesized compounds were evaluated for their inhibitory activity towards PARP-1 and examined for their anti-proliferative activity against MCF-7 and HCT116 human cancer cell lines. The synthesized compounds showed promising activity where compounds S2 and S7 emerged as the most potent PARP-1 inhibitors with an IC50 value of 4.06 ± 0.18 and 3.61 ± 0.15 nM, respectively compared to that of Olaparib 5.77 nM and high cytotoxicity against MCF-7 with IC50 2.65 ± 0.05 and 1.28 ± 1.12 μM, respectively (Staurosporine 7.258 μM). Compound S8 remarkably showed the highest cell growth inhibition against MCF-7 and HCT116 with an IC50 value of 0.66 ± 0.05 and 2.76 ± 0.06 μM, respectively. Furthermore, molecular docking of the compounds into the PARP-1 active site was performed to explore the probable binding mode. Finally, most of the synthesized compounds were predicted to have good pharmacokinetics properties in a theoretical kinetic study.

In continuation of our efforts to develop new antiproliferative agents that could be effective and selective in treatment of cancer, we designed and synthesized new hybrid structures containing an arylsulfonamide scaffold linked to a steroidal skeleton through a 1,2,3-triazole ring. Both in vitro cytotoxicity on breast MCF-7 cancer cells and human placental aromatase enzyme (pAROM) inhibition assays were performed on new hybrids. All new hybrids showed marked cytotoxic activity against breast MCF-7 cancer cells (IC50 = 3.56–43.76 μM) in comparison to staurosporine (IC50 = 4.06 μM). Tumor selectivity index was higher for some of the new hybrids on normal fibroblast (F-180) cells (TS = 1.5–25) in comparison to staurosporine (TS = 2.5). The p-nitro derivative exhibited the best inhibitory activity on the pAROM with an IC50 of 64.6 ng/cm3, compared to hybrids unsubstituted derivative, p-bromo derivative, and letrozole (IC50 = 375.14, 269.86, and 132.86 ng/cm3, respectively). Furthermore, the p-nitro hybrid arrested the cell cycle selectively at the G2/M phase, in addition to inducing both early and late apoptotic processes of breast MCF-7 cancer cells. Molecular docking studies were performed within pAROM to explore the binding modes of the new hybrids. Collectively, the antiproliferative profile of new hybrids indicates how good they are as promising leads for developing tumor-specific cytotoxins, and deserve further studies to optimize their structure and in vivo activity.

A new set of 4,6,7,8-tetrahydroquinolin-5(1H)-ones were designed as cytotoxic agents against breast cancer cell line (MCF-7) and synthesised under ultrasonic irradiation using chitosan decorated copper nanoparticles (CS/CuNPs) catalyst. The new compounds 4b, 4j, 4k, and 4e exhibited the most potent cytotoxic activity of IC50 values (0.002 − 0.004 µM) comparing to Staurosporine of IC50; 0.005 μM. The latter derivatives exhibited a promising safety profile against the normal human WI38 cells of IC50 range 0.0149 − 0.048 µM. Furthermore, the most promising cytotoxic compounds 4b, 4j were evaluated as multi-targeting agents against the RTK protein kinases; EGFR, HER-2, PDGFR-β, and VEGFR-2. Compound 4j showed promising inhibitory activity against HER-2 and PDGFR-β of IC50 values 0.17 × 10−3, 0.07 × 10−3 µM in comparison with the reference drug sorafenib of IC50; 0.28 × 10−3, 0.13 × 10−3 µM, respectively. In addition, 4j induced apoptotic effect and cell cycle arrest at G2/M phase preventing the mitotic cycle in MCF-7 cells., Graphical Abstract

We evaluated standard-of-care (SOC) treatment with or without midostaurin to prevent relapse following allogeneic hematopoietic stem cell transplant (alloHSCT) in patients with acute myeloid leukemia (AML) harboring internal tandem duplication (ITD) in FLT3. Adults (aged 18–70 years) who received alloHSCT in first complete remission, had achieved hematologic recovery, and were transfusion independent were randomized to receive SOC with or without midostaurin (50 mg twice daily) continuously in twelve 4-week cycles. The primary endpoint was relapse-free survival (RFS) 18 months post-alloHSCT. Sixty patients were randomized (30/arm); 30 completed all 12 cycles (midostaurin + SOC, n = 16; SOC, n = 14). The estimated 18-month RFS (95% CI) was 89% (69–96%) in the midostaurin arm and 76% (54–88%) in the SOC arm (hazard ratio, 0.46 [95% CI, 0.12–1.86]; P = 0.27); estimated relapse rates were 11% and 24%, respectively. Inhibition of FLT3 phosphorylation to FLT3-ITD AML. (ClinicalTrials.gov identifier: NCT01883362).

Upon exposure to osmotic stimuli, AQP1 is phosphorylated and translocates to the cell membrane. After the initial response, the Ca2+/CaMK2 pathway is activated to regulate membrane localization and the activity of AQP1, which leads to a change in cell size and adjustment to the environment. These changes are blocked by the calmodulin antagonist W‐7., The aquaporins (AQPs) are a family of integral membrane proteins which play critical roles in controlling transcellular water movement in various tissues throughout the body. AQP1 helps mediate the cellular response to osmotic stress and tissue water permeability. However, the mechanism by which AQP1 mediates changes in cell volume is not completely clear. Here, we investigated how AQP1 responds to and controls cell volume upon osmotic stimuli during the early phase after the immediate response. Cells overexpressing AQP1 were exposed to hypotonic or hypertonic medium in the presence or absence of staurosporine or W‐7 hydrochloride, and fluorescence imaging was performed at 0, 5, 10, and 15 min later. Osmotic stimuli induced redistribution of AQP1 into the cell membrane, hypotonic stimuli caused cell enlargement, and hypertonic stimuli induced a reduction in cell size, which was blocked by T157A/T239A mutations. Changes in cell size induced by osmotic stimuli were blocked by an antagonist of calmodulin kinase, W‐7 hydrochloride, but not by the PKC inhibitor staurosporine. These results suggest that calmodulin kinase regulates AQP1 activity during the early response to osmotic stimuli.

An important limitation of FLT3 tyrosine kinase inhibitors (TKIs) in FLT3-ITD positive AML is the development of resistance. To better understand resistance to FLT3 inhibition, we examined FLT3-ITD positive cell lines which had acquired resistance to midostaurin or sorafenib. In 6 out of 23 TKI resistant cell lines we were able to detect a JAK1 V658F mutation, a mutation that led to reactivation of the CSF2RB-STAT5 pathway. Knockdown of JAK1, or treatment with a JAK inhibitor, resensitized cells to FLT3 inhibition. Out of 136 patients with FLT3-ITD mutated AML and exposed to FLT3 inhibitor, we found seven different JAK family mutations in six of the cases (4.4%), including five bona fide, activating mutations. Except for one patient, the JAK mutations occurred de novo (n = 4) or displayed increasing variant allele frequency after exposure to FLT3 TKI (n = 1). In vitro each of the five activating variants were found to induce resistance to FLT3-ITD inhibition, which was then overcome by dual FLT3/JAK inhibition. In conclusion, our data characterize a novel mechanism of resistance to FLT3-ITD inhibition and may offer a potential therapy, using dual JAK and FLT3 inhibition.

Chinese hamster ovary (CHO) cells are used as host cells for biopharmaceutical production, including monoclonal antibodies (mAbs). Arresting the cell cycle with chemical compounds is an effective approach to improve biopharmaceutical productivity. In a previous study, potential new cell cycle-arresting compounds were screened from marine-derived microorganism culture extracts, and it was suggested that staurosporine might improve mAb productivity in CHO cells via cell cycle arrest. The purpose of this study was to demonstrate the effectiveness of staurosporine as a cell-cycle arresting compound to improve mAb productivity. The optimal staurosporine concentration range was initially investigated using batch cultures. Thereafter, the effects on the culture profile and mAb productivity were evaluated using fed-batch cultures. Staurosporine at concentrations ≥10 nM induced cell death, but at concentrations ≤5 nM did not. In the range of 2-4 nM, cell growth was inhibited, whereas the specific production rate (Qp) and cell longevity were improved in a dose-dependent manner. The Qp and maximum mAb concentration with 4 nM staurosporine improved by 36.3 and 5.2%, respectively, compared to those with control conditions. Cell viability post-culture without staurosporine was 40.0 ± 0.3%, whereas with 4 nM staurosporine, it was 90.1 ± 1.0%. Flow cytometric analysis indicated cell-cycle arrest at the G1/G0 phase with 4 nM staurosporine addition. The present study highlighted the efficacy of staurosporine in improving mAb production by causing cell-cycle arrest. Further research into staurosporine analogs and how to use them will lead to development of more effective industrial production technologies of biopharmaceuticals.

Due to the ever-expanding functions attributed to autophagy, there is widespread interest in understanding its contribution to human physiology; however, its specific cellular role as a stress-response mechanism is still poorly defined. To investigate autophagy's role in this regard, we repeatedly subjected cultured mouse myoblasts to two stresses with diverse impacts on autophagic flux: amino acid and serum withdrawal (Hank's balanced salt solution [HBSS]), which robustly induces autophagy, or low-level toxic stress (staurosporine, STS). We found that intermittent STS (int-STS) administration caused cell cycle arrest, development of enlarged and misshapen cells/nuclei, increased senescence-associated heterochromatic foci and senescence-associated β-galactosidase activity, and prevented myogenic differentiation. These features were not observed in cells intermittently incubated in HBSS (int-HB). While int-STS cells displayed less DNA damage (phosphorylated H2A histone family, member X content) and caspase activity when administered cisplatin, int-HB cells were protected from STS-induced cell death. Interestingly, STS-induced senescence was attenuated in autophagy related 7-deficient cells. Therefore, while repeated nutrient withdrawal did not cause senescence, autophagy was required for senescence caused by toxic stress. These results illustrate the context-dependent effects of different stressors, potentially highlighting autophagy as a distinguishing factor.

Midostaurin is an FMS-like tyrosine kinase 3 receptor (FLT3) inhibitor that provides renewed hope for treating acute myeloid leukaemia (AML). The limited efficacy of this compound as a monotherapy contrasts with that of its synergistic combination with standard cytarabine and daunorubicin (Dau), suggesting a therapeutic benefit that is not driven only by FLT3 inhibition. In an AML context, the activity of the enzyme aldo-keto reductase 1C3 (AKR1C3) is a crucial factor in chemotherapy resistance, as it mediates the intracellular transformation of anthracyclines to less active hydroxy metabolites. Here, we report that midostaurin is a potent inhibitor of Dau inactivation mediated by AKR1C3 in both its recombinant form as well as during its overexpression in a transfected cell model. Likewise, in the FLT3- AML cell line KG1a, midostaurin was able to increase the cellular accumulation of Dau and significantly decrease its metabolism by AKR1C3 simultaneously. The combination of those mechanisms increased the nuclear localization of Dau, thus synergizing its cytotoxic effects on KG1a cells. Our results provide new in vitro evidence of how the therapeutic activity of midostaurin could operate beyond targeting the FLT3 receptor.

The canonical function of Bcl-2 family proteins is to regulate mitochondrial membrane integrity. In response to apoptotic signals the multi-domain pro-apoptotic proteins Bax and Bak are activated and perforate the mitochondrial outer membrane by a mechanism which is inhibited by their interaction with pro-survival members of the family. However, other studies have shown that Bax and Bak may have additional, non-canonical functions, which include stress-induced nuclear envelope rupture and discharge of nuclear proteins into the cytosol. We show here that the apoptotic stimuli cisplatin and staurosporine induce a Bax/Bak-dependent degradation and subcellular redistribution of nesprin-1 and nesprin-2 but not nesprin-3, of the linker of nucleoskeleton and cytoskeleton (LINC) complex. The degradation and redistribution were caspase-independent and did not occur in Bax/Bak double knockout (DKO) mouse embryo fibroblasts (MEFs). Re-expression of Bax in Bax/Bak DKO MEFs restored stress-induced redistribution of nesprin-2 by a mechanism which requires Bax membrane localization and integrity of the α helices 5/6, and the Bcl-2 homology 3 (BH3) domain. We found that nesprin-2 interacts with Bax in close proximity to perinuclear mitochondria in mouse and human cells. This interaction requires the mitochondrial targeting and N-terminal region but not the BH3 domain of Bax. Our results identify nesprin-2 as a Bax binding partner and also a new function of Bax in impairing the integrity of the LINC complex.

2,3,5,4'-Tetrahydroxystilbene-2-O-β-d-glucoside (THSG) is the major active ingredient in Plygonum multiflorum that displays a great deal of health-benefits including anti-oxidation, anti-hyperlipidemia, anti-cancer, anti-inflammation and neuroprotection. However, it is unclear whether THSG exerts neuroprotective functions by regulating neurotrophic factors and their associated signaling pathways. In this study, hippocampal neurons were challenged with staurosporine (STS) to establish a neural damage model. We found that STS-induced cytotoxicity introduced significant morphological collapse and initiating cell apoptosis, along with the down regulation of BDNF and TrkB/Akt signaling axis. In contrast, neurons pretreated with THSG showed resistance to STS-induced toxicity and maintained cell survival. THSG rescued STS induced dysfunctions of BDNF and its associated TrkB/Akt signaling, and restored the expression of Bcl-2 and Caspase-3. However, inhibition of TrkB activity by K252a or Akt signaling by LY294002 abolished the neuroprotective effects of THSG. Therefore, BDNF and TrkB/Akt signaling axis is a promise target for THSG mediated neuroprotective functions.

3D cell culture models have been developed to better mimic the physiological environments that exist in human diseases. As such, these models are advantageous over traditional 2D cultures for screening drug compounds. However, the practicalities of transitioning from 2D to 3D drug treatment studies pose challenges with respect to analysis methods. Patient-derived tumor organoids (PDTOs) possess unique features given their heterogeneity in size, shape, and growth patterns. A detailed assessment of the length scale at which PDTOs should be evaluated (i.e., individual cell or organoid-level analysis) has not been done to our knowledge. Therefore, using dynamic confocal live cell imaging and data analysis methods we examined tumor cell growth rates and drug response behaviors in colorectal cancer (CRC) PDTOs. High-resolution imaging of H2B-GFP-labeled organoids with DRAQ7 vital dye permitted tracking of cellular changes, such as cell birth and death events, in individual organoids. From these same images, we measured morphological features of the 3D objects, including volume, sphericity, and ellipticity. Sphericity and ellipticity were used to evaluate intra- and interpatient tumor organoid heterogeneity. We found a strong correlation between organoid live cell number and volume. Linear growth rate calculations based on volume or live cell counts were used to determine differential responses to therapeutic interventions. We showed that this approach can detect different types of drug effects (cytotoxic vs cytostatic) in PDTO cultures. Overall, our imaging-based quantification workflow results in multiple parameters that can provide patient- and drug-specific information for screening applications.

Oncoprotein E6 of high-risk human papillomavirus (HPV) plays a critical role in inducing cell immortalization and malignancy. E6 downregulates caspase-dependent pathway through the degradation of p53. However, the effect of HPV E6 on other pathways is still under investigation. In the present study, we found that HPV E6 directly binds to all three forms (precursor, mature, and apoptotic) of apoptosis-inducing factor (AIF) and co-localizes with apoptotic AIF. This binding induced MG132-sensitive reduction of AIF expression in the presence of E6 derived from HPV16 (16E6), a cancer-causing type of HPV. Conversely, E6 derived from a non-cancer-causing type of HPV, HPV6 (6E6), did not reduce the levels of AIF despite its interaction with AIF. Flow cytometric analysis revealed that 16E6, but not 6E6, suppressed apoptotic AIF-induced chromatin degradation (an indicator of caspase-independent apoptosis) and staurosporine (STS, a protein kinase inhibitor)-induced apoptosis. AIF knockdown reduced STS-induced apoptosis in both of 16E6-expressing and 6E6-expressing cells; however, the reduction in 16E6-expressing cells was lower than that in 6E6-expressing cells. These findings indicate that 16E6, but not 6E6, blocks AIF-mediated apoptosis, and that AIF may represent a novel therapeutic target for HPV-induced cervical cancer., 論文

Monoclonal antibodies (mAbs) are active pharmaceutical ingredients in antibody drugs, produced mainly using recombinant Chinese hamster ovary (CHO) cells. The regulation of recombinant CHO cell proliferation can improve the productivity of heterologous proteins. Chemical compound approaches for cell cycle regulation have the advantages of simplicity and ease of use in industrial processes. However, CHO cells have genetic and phenotypic diversity, and the effects of such compounds might depend on cell line and culture conditions. Increasing the variety of cell cycle inhibitors is a promising strategy to overcome the dependency. Marine microorganisms are a vast and largely undeveloped source of secondary metabolites with physiological activity. In this study, we focused on secondary metabolites of marine microorganisms and evaluated their effectiveness as cell cycle inhibitory compounds. Of 720 extracts from microorganisms (400 actinomycetes and 320 filamentous fungi) collected from the Okinawan Sea, we identified nine extracts that decreased the specific growth rate and increased the specific production rate without reducing cell viability. After fractionating the extracts, the components of active fractions were estimated using time-of-flight mass spectrometry analysis. Then, four compounds, including staurosporine and undecylprodigiosin were deduced to be active compounds. These compounds have been reported to exert a cell cycle inhibitory effect on mammalian cells. These compounds might serve as additives to improve mAb production in CHO cells. This study indicates that secondary metabolites of marine microorganisms are a useful source for new cell cycle inhibitory compounds that can increase mAb production in CHO cells.

With the advent of new targeted drugs in hematology and oncology patient prognosis is improved. Combination with antifungal prophylaxis challenges clinicians due to pharmacological profiles prone to drug–drug interactions (DDI). Midostaurin is a novel agent for FLT3-TKD/-ITDmut-acute myeloid leukemia (AML) and metabolized via cytochrome P450 3A4 (CYP3A4). Posaconazole is a standard of care antifungal agent used for prophylaxis during induction treatment of AML and a strong CYP3A4 inhibitor. Concomitant administration of both drugs leads to elevated midostaurin exposure. Both drugs improve overall survival at low numbers needed to treat. The impact of CYP3A4-related DDI remains to be determined. Severe adverse events have been observed; however, it remains unclear if they can be directly linked to DDI. The lack of prospective clinical studies assessing incidence of invasive fungal infections and clinical impact of DDI contributes to neglecting live-saving antifungal prophylaxis. Management strategies to combine both drugs have been proposed, but evidence on which approach to use is scarce. In this review, we discuss several approaches in the specific clinical setting of concomitant administration of midostaurin and posaconazole and give examples from everyday clinical practice. Therapeutic drug monitoring will become increasingly important to individualize and personalize antineoplastic concomitant and antifungal treatment in the context of DDI. Pharmaceutical companies addressing the issue in clinical trials may take a pioneer role in this field. Other recently developed and approved drugs for the treatment of AML likely inhere potential of DDI marking a foreseeable issue in future treatment of this life-threatening disease.

Previous efforts to determine whether or not the transcription factor and tumor suppressor protein p53 is required for DNA damage-induced apoptosis in pluripotent embryonic stem cells (ESCs) produced contradictory conclusions. To resolve this issue, p53+/+ and p53−/− ESCs derived by two different methods were used to quantify time-dependent changes in nuclear DNA content; annexin-V binding; cell permeabilization; and protein expression, modification, and localization. The results revealed that doxorubicin (Adriamycin [ADR]) concentrations 10 to 40 times less than commonly used in previous studies induced the DNA damage-dependent G2-checkpoint and completed apoptosis within the same time frame, regardless of the presence or absence of p53, p21, and PUMA. Increased ADR concentrations delayed initiation of apoptosis in p53−/− ESCs, but the rates of apoptosis remained equivalent. Similar results were obtained by inducing apoptosis with either staurosporine inhibition of kinase activities or WX8 disruption of lysosome homeostasis. Differentiation of ESCs by LIF deprivation revealed p53-dependent formation of haploid cells, increased genomic stability, and suppression of the G2-checkpoint. Minimal induction of DNA damage now resulted in p53-facilitated apoptosis, but regulation of pluripotent gene expression remained p53-independent. Primary embryonic fibroblasts underwent p53-dependent total cell cycle arrest (a prelude to cell senescence), and p53-independent apoptosis occurred in the presence of 10-fold higher levels of ADR, consistent with previous studies. Taken together, these results reveal that the multiple roles of p53 in cell cycle regulation and apoptosis are first acquired during pluripotent stem cell differentiation.

Background/aims Sodium is a key player in the fundamental cell functions. Fluorescent probes are indispensable tools for monitoring intracellular sodium levels in single living cells. Since the fluorescence of sodium-sensitive dyes in cells is significantly different from that in an aqueous solution, the fluorescence signal is calibrated in situ indirectly using ionophores for equalizing external and intracellular ion concentration. Attempts to compare data obtained using fluorescent probes and by direct flame emission analysis are sparse and results are inaccurate. Methods We determined the intracellular sodium concentration in U937 cells by flow cytometry using the Na+-sensitive probe Asante Natrium Green-2 (ANG), and by standard flame emission photometry combined with the cellular water determination by cell density in Percoll gradient. The intracellular Na+ concentrations was modified using known ionophores or, alternatively, by blocking the sodium pump with ouabain or by causing cell apoptosis with staurosporine. Results It is revealed that both methods are comparable when intracellular sodium concentration was modified by ouabain-mediated blockage of the sodium pump or staurosporine-induced apoptosis. The ANG fluorescence of cells treated with ionophores is approximately two times lower than that in cells with the same Na+ concentration but not treated with ionophores. Although the mechanism is still unknown, this effect should be taken into account when a quantitative assessment of the concentration of intracellular sodium is required. Conclusion The sodium sensitive dye ANG-2 is a sensitive and useful probe for determination changes in Na+ content and concentration both in single cells and subcellular microparticles. The ANG fluorescence determined in the studied cells in the absence of ionophores, cannot be used as a measure of the real intracellular concentration of Na+ if calibration was carried out in the presence of ionophores.

Systemic mastocytosis (SM) has greatly benefited from the broad application of precision medicine techniques to hematolymphoid neoplasms. Sensitive detection of the recurrent KIT D816V mutation and use of next-generation sequencing (NGS) panels to profile the genetic landscape of SM variants have been critical adjuncts to the diagnosis and subclassification of SM, and development of clinical-molecular prognostic scoring systems. Multilineage KIT involvement and multimutated clones are characteristic of advanced SM (advSM), especially SM with an associated hematologic neoplasm (AHN). A major challenge is how to integrate conventional markers of mast cell disease burden (percentage of bone marrow mast cell infiltration and serum tryptase levels) with molecular data (serial monitoring of both KIT D816V variant allele frequency and NGS panels) to lend more diagnostic and prognostic clarity to the heterogeneous clinical presentations and natural histories of advSM. The approval of the multikinase/KIT inhibitor midostaurin has validated the paradigm of KIT inhibition in advSM, and the efficacy and safety of second-generation agents, such as the switch-control inhibitor ripretinib (DCC-2618) and the D816V-selective inhibitor avapritinib (BLU-285) are being further defined in ongoing clinical trials. Looking forward, perhaps the most fruitful marriage of the advances in molecular genetics and treatment will be the design of adaptive basket trials that combine histopathology and genetic profiling to individualize treatment approaches for patients with diverse AHNs and relapsed/refractory SM.

FLT3‐ITD tyrosine kinase inhibitors (TKI) show limited clinical activity in acute myeloid leukemia (AML) due to emerging resistance. TKI resistance is mediated by secondary FLT3‐ITD mutations only in a minority of cases. We hypothesize that the cytokine CCL5 protects AML cells from TKI‐mediated cell death and contributes to treatment resistance. We generated PKC412‐ and sorafenib‐resistant MOLM‐13 cell lines as an in vitro model to study TKI resistance in AML. Increased CCL5 levels were detected in supernatants from PKC412‐resistant cell lines compared to TKI‐sensitive cells. Moreover, CCL5 treatment of TKI‐sensitive cells induced resistance to PKC412. In resistant cell lines with high CCL5 release, we observed a significant downregulation of the CCL5‐receptor CCR5 and CXCR4. In these cell lines, TKI resistance could be partly overcome by addition of the CXCR4‐receptor antagonist plerixafor. Microarray and intracellular flow cytometry analyses revealed increased p‐Akt or p‐Stat5 levels in PKC412‐resistant cell lines releasing high amounts of CCL5. Treatment with the CXCR4 antagonist plerixafor, αCCL5, or CCR5‐targeting siRNA led to a decrease of p‐Akt‐positive cells. Transient transfection of sensitive MOLM‐13 cells with a CCL5‐encoding vector mediated resistance against PKC412 and led to an increase in p‐Akt‐positive and p‐Stat5‐positive cells. Isolated AML blasts from patients treated with PKC412 revealed that CCL5 transcript levels increase significantly at relapse. Taken together, our findings indicate that CCL5 mediates resistance to FLT3‐TKIs in FLT3‐ITD‐mutated AML and could possibly serve as a biomarker to predict drug resistance., Increased signaling of the CCL5‐CCR5 pathway can protect FLT3‐ITD+ AML cells from cell death mediated by tyrosine kinase inhibitors like midostaurin. Thereby, either increased CCL5 release or upregulation of the CCR5/CXCR4 receptors induces enhanced levels of p‐AKT, p‐STAT5, and Bcl‐2, possibly mediating enhanced survival. Therefore, CCL5 could pave the way for new treatment strategies in FLT3‐ITD+ AML patients at relapse.

Kinase-targeted therapy has been widely used as a lifesaving strategy for cancer patients. However, many patients treated with targeted cancer drugs are clinically observed to rapidly develop acquired resistance. Kinase gatekeeper mutation is one of the most chief factors contributing to the resistance, which modulates the accessibility of kinase's ATP-binding pocket. Previously, the pan-kinase inhibitor Staurosporine and its analogs (termed as Staralogs) have been reported to exhibit wild-type sparing selectivity for some kinase gatekeeper mutants, such as EGFR T790M, Her2 T798M and cSrc T338M. Here, we describe an integrative approach to systematically profile the molecular response of 15 representative Staralogs to 17 kinase gatekeeper mutations in targeted cancer therapy. With the profile we are able to divide gatekeeper mutations into three classes (i.e. classes I, II and III) and to divide Staralogs into two groups (i.e. groups 1 and 2) using heuristic clustering. The class I and II mutations confer consistent sensitivity and resistance for all Staralogs, respectively, while the class III mutations address divergent effects on different Staralogs. The mutations to Ile residue can generally reduce Staralog affinity by inducing unfavorable steric hindrance, whereas the mutations to Met and Leu residues would improve Staralog affinity by establishing favorable S···π interaction, van der Waals packing and/or hydrophobic contact. The group 1 and 2 Staralogs are primarily determined by carbonyl or hydroxyl substitution state at the position 7 of Staralog core, where points to kinase gatekeeper residue and can thus be directly influenced by gatekeeper mutation.

Dysregulation of PARP10 has been implicated in various tumor types and plays a vital role in delaying hepatocellular carcinoma (HCC) progression. However, the mechanisms controlling the expression and activity of PARP10 in HCC remain mostly unknown. The crosstalk between PLK1, PARP10, and NF-κB pathway in HCC was determined by performing different in vitro and in vivo assays, including mass spectrometry, kinase, MARylation, chromatin immunoprecipitation, and luciferase reporter measurements. Functional examination was performed by using small chemical drug, cell culture, and mice HCC models. Correlation between PLK1, NF-κB, and PARP10 expression was determined by analyzing clinical samples of HCC patients with using immunohistochemistry. PLK1, an important regulator for cell mitosis, directly interacts with and phosphorylates PARP10 at T601. PARP10 phosphorylation at T601 significantly decreases its binding to NEMO and disrupts its inhibition to NEMO ubiquitination, thereby enhancing the transcription activity of NF-κB toward multiple target genes and promoting HCC development. In turn, NF-κB transcriptionally inhibits the PARP10 promoter activity and leads to its downregulation in HCC. Interestingly, PLK1 is mono-ADP-ribosylated by PARP10 and the MARylation of PLK1 significantly inhibits its kinase activity and oncogenic function in HCC. Clinically, the expression levels of PLK1 and phosphor-p65 show an inverse correlation with PARP10 expression in human HCC tissues. These findings are the first to uncover a PLK1/PARP10/NF-κB signaling circuit that underlies tumorigenesis and validate PLK1 inhibitors, alone or with NF-κB antagonists, as potential effective therapeutics for PARP10-expressing HCC.

Assessing the expression of channels on the cell membrane is a necessary step in studying the functioning of ion channels in living cells. We explore, first, if endogenous VRAC can be assayed using flow cytometry and a commercially available antibody against an extracellular loop of the LRRC8A, also known as SWELL1, subunit of the VRAC channel. The second goal is to determine if an increase in the number of VRAC channels at the cell membrane is responsible for an increase in chloride permeability of the membrane in two well-known cases: during staurosporine (STS)-induced apoptosis and after water balance disturbance caused by hypotonic medium. Human suspension lymphoid cells U937 were used as they are suitable for flow fluorometry and because we have recently studied their membrane chloride permeability during apoptosis. We found that surface expression of endogenous LRRC8A subunits can be quantified in living U937 cells using flow fluorometry with the Alomone Lab antibody. Further, we revealed that treatment of cells for 1 hour using STS or a hypotonic solution did not change the number of LRRC8A subunits to the extent that would correspond to changes in the membrane chloride permeability determined by ion content analysis. This indicates that prolonged increase in chloride permeability of the cell membrane during apoptotic cell shrinkage or cell volume regulation under hypotonicity in U937 cells occurs without altering cell surface expression of VRAC.

Recently, several targeted agents have been developed for specific subsets of patients with acute myeloid leukaemia (AML), including midostaurin, the first FDA‐approved FLT3 inhibitor for newly diagnosed patients with FLT3 mutations. However, in the initial Phase I/II clinical trials, some patients without FLT3 mutations had transient responses to midostaurin, suggesting that this multi‐targeted kinase inhibitor might benefit AML patients more broadly. Here, we demonstrate submicromolar efficacy of midostaurin in vitro and efficacy in vivo against wild‐type (wt) FLT3‐expressing AML cell lines and primary cells, and we compare its effectiveness with that of other FLT3 inhibitors currently in clinical trials. Midostaurin was found to synergize with standard chemotherapeutic drugs and some targeted agents against AML cells without mutations in FLT3. The mechanism may involve, in part, the unique kinase profile of midostaurin that includes proteins implicated in AML transformation, such as SYK or KIT, or inhibition of ERK pathway or proviability signalling. Our findings support further investigation of midostaurin as a chemosensitizing agent in AML patients without FLT3 mutations.

We present a 61-year-old male with FLT3-mutated acute myeloid leukemia treated with midostaurin who developed acute kidney injury requiring hemodialysis and pulmonary renal syndrome. Antibodies to proteinase-3, myeloperoxidase, and glomerular basement membrane were negative. Renal biopsy confirmed acute pauci-immune focal necrotizing glomerulonephritis (GN) with fibrin crescents indicating rapidly progressing glomerulonephritis. He improved with pulse methylprednisolone, intravenous cyclophosphamide, and plasma exchange with resolution of hemoptysis. This case highlights the importance of prompt renal biopsy to guide early initiation of life-saving therapies. To our knowledge, this is the first reported case of ANCA-negative pauci-immune necrotizing GN likely secondary to midostaurin.

Mutations in the E3 ubiquitin ligase CBL, found in several myeloid neoplasms, lead to decreased ubiquitin ligase activity. In murine systems, these mutations are associated with cytokine‐independent proliferation, thought to result from the activation of hematopoietic growth receptors, including FLT3 and KIT. Using cell lines and primary patient cells, we compared the activity of a panel of FLT3 inhibitors currently being used or tested in AML patients and also evaluated the effects of inhibition of the non‐receptor tyrosine kinase, SYK. We show that FLT3 inhibitors ranging from promiscuous to highly targeted are potent inhibitors of growth of leukaemia cells expressing mutant CBL in vitro, and we demonstrate in vivo efficacy of midostaurin using mouse models of mutant CBL. Potentiation of effects of targeted FLT3 inhibition by SYK inhibition has been demonstrated in models of mutant FLT3‐positive AML and AML characterized by hyperactivated SYK. Here, we show that targeted SYK inhibition similarly enhances the effects of midostaurin and other FLT3 inhibitors against mutant CBL‐positive leukaemia. Taken together, our results support the notion that mutant CBL‐expressing myeloid leukaemias are highly sensitive to available FLT3 inhibitors and that this effect can be significantly augmented by optimum inhibition of SYK kinase.

The FMS-like tyrosine kinase 3 (FLT3) gene is mutated in approximately one third of patients with acute myeloid leukemia (AML), either by internal tandem duplications (FLT3-ITD), or by a point mutation mainly involving the tyrosine kinase domain (FLT3-TKD). Patients with FLT3-ITD have a high risk of relapse and low cure rates. Several FLT3 tyrosine kinase inhibitors have been developed in the last few years with variable kinase inhibitory properties, pharmacokinetics, and toxicity profiles. FLT3 inhibitors are divided into first generation multi-kinase inhibitors (such as sorafenib, lestaurtinib, midostaurin) and next generation inhibitors (such as quizartinib, crenolanib, gilteritinib) based on their potency and specificity of FLT3 inhibition. These diverse FLT3 inhibitors have been evaluated in myriad clinical trials as monotherapy or in combination with conventional chemotherapy or hypomethylating agents and in various settings, including front-line, relapsed or refractory disease, and maintenance therapy after consolidation chemotherapy or allogeneic stem cell transplantation. In this practical question-and-answer-based review, the main issues faced by the leukemia specialists on the use of FLT3 inhibitors in AML are addressed.

The Na+/K+-ATPase, present in the basolateral membrane of human corneal endothelial cells (HCECs), is known to play an important role for corneal transparency. Na+/K+-ATPase dysfunction is one of the major causes of corneal decompensation. The ethanol extract of Diospyros kaki (EEDK) has been reported to increase corneal cell viability. Thus, we treated HCECs with EEDK and studied its effects on HCECs survival and Na+/K+-ATPase against cytotoxic drugs like staurosporine (ST) and ouabain (OU). Firstly, survival assays, (MTT assay and live dead-imaging) showed that decreased HCECs viability by ST and OU was significantly recovered by EEDK co-treatment. Secondly, Na+/K+-ATPase activity assays revealed that EEDK enhanced Na+/K+-ATPase enzymatic activity (* p < 0.01) with/without ST and OU. Finally, Na+/K+-ATPase expression analysis (Western Blot and confocal microscopy) demonstrated that EEDK treatment with/without ST and OU facilitates Na+/K+-ATPase expression in HCECs. Taken together, our findings led us to the conclusion that EEDK might aid HCECs survival in vitro by increasing the activity and expression of Na+/K+-ATPase enzyme. Since Na+/K+-ATPase activity is important to maintain cellular function of HCECs, we suggest that EEDK can be a potential effective agent against corneal edema and related corneal disorders.

Neuroblastomas are one of the most common types of solid tumors in infants and children and are responsible for approximately 15% of childhood cancer deaths. Neuroblastomas rarely have mutations in p53, with less than 2% of NB containing mutations in p53, compared to up to 60% for other tumor classes. Previous studies on the therapeutic combination of a DNA damaging agent and checkpoint kinase 1 (Chk1) inhibitor have shown that DNA damage-induced cell cycle arrest can be specifically abrogated in p53-defective tumors. However, some p53-wildtype tumors have also been shown to be sensitive to this therapeutic combination, suggesting that these cells have other defects in the p53 response that can be exploited for therapeutic purposes. In the current study, we investigated the response to the combination of a DNA damaging agent (SN38) and a Chk1 inhibitor (UCN-01) of four p53-wildtype neuroblastoma cell lines: SK-N-SH, SH-SY5Y, SK-N-AS, and Lan-5. When the cells were treated with concentrations of SN38 ranging from 0–30 ng/ml, all four cell lines accumulated p53 which was phosphorylated on serines 15 and 20. However, only the SK-N-SH were found to activate p21waf1 and repress cyclin B. In order to assess sensitivity to UCN-01-mediated abrogation of cell cycle arrest, cell were treated with 10 ng/ml SN38 for 24 h, followed by 25 nM UCN-01 for 6 and 24 h. The SK-N-SH showed no sensitivity to UCN-01 treatment whereas the SH-SY5Y, SK-N-AS, and Lan-5 abrogated G2 arrest within 24 h. Our recent studies revealed that cells that are sensitive to checkpoint abrogation lack p53 dimers and tetramers, so we analyzed the oligomerization status of p53 in all four cell lines using glutaraldehyde crosslinking. The SK-N-SH cells possessed levels of p53 dimers and tetramers similar to what has previously been reported in p53-wildtype MCF10A cells. The SH-SY5Y, SK-N-AS, and Lan-5 however, had extremely low to undetectable levels of dimers and tetramers. Our study also showed no cytoplasmic accumulation of p53 in these cells contrary to some previous reports. The results of this study suggest that oligomerization status may serve as an indicator of sensitivity of p53-wildtype tumors to the therapeutic combination of DNA damaging agent and Chk1 inhibitor.

Background FMS-like tyrosine kinase-3 (FLT3-ITD) mutations are some of the most common mutations in acute myeloid leukemia (AML), and patient outcomes have improved since the advent of tyrosine kinase inhibitors. First, granulocytic differentiation was described in FLT3-positive AML treated with FLT3 inhibitors, and more recently, monocytic differentiation was reported. Methods Two patients with myelomonocytic cells in their bone marrow were identified during routine follow-up after AML treatment that included FLT3 inhibitors. The bone marrow study was done as standard of care. Results Both patients had FLT3-ITD+ AML and showed an atypical maturing monocytic cell population and a decrease in the leukemic blast cell population after FLT3 inhibitor therapy. Concurrent genetic testing revealed persistent genetic abnormalities. Conclusions These cases illustrate monocytic maturation in FLT3+ AML after FLT3 inhibitor treatment. It is critical for pathologists and clinicians to be aware of the differentiation phenomenon, as these patients have persistent molecular abnormalities despite response to treatment and normalization of blast counts.

Glutamate-induced excitotoxicity plays a critical role in the neurological impairment caused by middle cerebral artery occlusion. Achyranthes bidentata polypeptides have been shown to protect against neurological functional damage caused by middle cerebral artery occlusion, but the underlying neuroprotective mechanisms and the relationship to glutamate-induced excitotoxicity remain unclear. Therefore, in the current study, we investigated the protective effects of Achyranthes bidentata polypeptides against glutamate-induced excitotoxicity in cultured hippocampal neurons. Hippocampal neurons were treated with Mg2+-free extracellular solution containing glutamate (300 µM) for 3 hours as a model of glutamate-mediated excitotoxicity (glutamate group). In the normal group, hippocampal neurons were incubated in Mg2+-free extracellular solution. In the Achyranthes bidentata polypeptide group, hippocampal neurons were incubated in Mg2+-free extracellular solution containing glutamate (300 µM) and Achyranthes bidentata polypeptide at different concentrations. At 24 hours after exposure to the agents, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and Hoechst 33258 staining were used to assess neuronal viability and nuclear morphology, respectively. Caspase-3 expression and activity were evaluated using western blot assay and colorimetric enzymatic assay, respectively. At various time points after glutamate treatment, reactive oxygen species in cells were detected by H2DCF-DA, and mitochondrial membrane potential was detected by rhodamine 123 staining. To examine the effect of Achyranthes bidentata polypeptides on glutamate receptors, electrophysiological recording was used to measure the glutamate-induced inward current in cultured hippocampal neurons. Achyranthes bidentata polypeptide decreased the percentage of apoptotic cells and reduced the changes in caspase-3 expression and activity induced by glutamate. In addition, Achyranthes bidentata polypeptide attenuated the amplitude of the glutamate-induced current. Furthermore, the glutamate-induced increase in intracellular reactive oxygen species and reduction in mitochondrial membrane potential were attenuated by Achyranthes bidentata polypeptide treatment. These findings collectively suggest that Achyranthes bidentata polypeptides exert a neuroprotective effect in cultured hippocampal neurons by suppressing the overactivation of glutamate receptors and inhibiting the caspase-3-dependent mitochondrial apoptotic pathway. All animal studies were approved by the Animal Care and Use Committee, Nantong University, China (approval No. 20120216-001) on February 16, 2012.

The α1-acid glycoprotein (AGP) is an abundant blood plasma protein with important immunomodulatory functions coupled to endogenous and exogenous ligand binding properties. Its affinity for many drug-like structures, however, means AGP can have a significant effect on the pharmokinetics and pharmacodynamics of numerous small molecule therapeutics. Staurosporine, and its hydroxylated forms UCN-01 and UCN-02, are kinase inhibitors that have been investigated at length as anti-tumour compounds. Despite their potency, these compounds display poor pharmokinetics due to binding to both AGP variants, AGP1 and AGP2. Recent renewed interest in UCN-01 as a cytostatic protective agent prompted us to solve the structure of the AGP2/UCN-01 complex by X-ray crystallography, revealing for the first time the precise binding mode of UCN-01. Solution NMR suggests AGP2 undergoes a significant conformational change upon ligand binding, but also that it uses a common set of sidechains with which it captures key groups of UCN-01 and other small molecule ligands. We anticipate that this structure and supporting NMR data will facilitate rational re-design of small molecules that could evade AGP and therefore improve tissue distribution.

Extracellular pH (pHe) is an important chemical factor in many cellular processes and disease pathologies. The routine sampling of pHe in vitro could lead to innovative advances in therapeutics. To this end, we have fabricated a novel gold-coated polymer mesh, which facilitates the real-time measurement of pHe via surface-enhanced Raman scattering (SERS). In this proof of concept study, we apply our SERS sensor to measure metabolically induced changes in the pHe of carcinoma-derived cell line HepG2/C3A. We demonstrate that gold-coated polyurethane electrospun nanofibers (AuNF) have strong and reproducible SERS spectra of surface-adsorbed analytes. By functionalizing AuNF with pH-responsive reporter 4-mercaptobenzoic acid (MBA), we have developed an accurate pH SERS sensor for the extracellular microenvironment. We cultured HepG2/C3A on the surface of MBA-AuNF and measured an acidic shift in pHe at the cell-fiber interface. Following exposure to staurosporine, an apoptosis-inducing drug, we observed changes in the HepG2/C3A cellular morphology indicative of controlled cell death, and detected an increase in the pHe of HepG2/C3A. These results demonstrate how subtle changes in pHe, induced by the metabolic activity of cells, can be measured with our novel SERS sensor MBA-AuNF. The excellent pH measurement performance of MBA-AuNF provides a unique platform to study extracellular pH on the microscale and will help to deepen our understanding of pHe in disease pathology.

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein which mediates staurosporine (STS) – induced cell death. AIF cleavage and translocation to the cytosol is thought to be calpain-1-dependent as calpain inhibitors reduce AIF proteolysis; however, many calpain inhibitors also inhibit matrix metalloproteinase-2 (MMP-2) activity, an intracellular and extracellular protease implicated in apoptosis. Here we investigated whether MMP-2 activity is affected in response to STS and if it contributes to AIF cleavage. Human fibrosarcoma HT1080 cells were treated with STS (0.1 µM, 0.25–24 h). A significant increase in cellular MMP-2 activity was seen by gelatin zymography after a 6 h STS treatment, prior to induction of cell necrosis. Western blot showed the time-dependent appearance of two forms of AIF (∼60 and 45 kDa) in the cytosol which were significantly increased at 6 h. Surprisingly, knocking down MMP-2 or inhibiting its activity with MMP-2 preferring inhibitors ARP-100 or ONO-4817, or inhibiting calpain activity with ALLM or PD150606, did not prevent the STS-induced increase in cytosolic AIF. These results show that although STS rapidly increases MMP-2 activity, the cytosolic release of AIF may be independent of the proteolytic activities of MMP-2 or calpain.