Chemoresistance is emerging as a major barrier to successful treatment in acute myeloid leukemia (AML), and bone marrow stromal cells (BMSCs) protect leukemia cells from chemotherapy eventually leading to recurrence. This study was designed to investigate the role of p21-activated kinase 1 (PAK1) in AML progression and chemosensitivity, highlighting the mechanism of stroma-mediated chemoresistance.in vitro, and co-culture model was established to investigate the role of PAK1 in BMSC-mediated drug resistance.The GEPIA and TCGA datasets were used to analyze the relationship between PAK1 mRNA expression and various clinical parameters of AML patients. Cell proliferation and apoptosis were examined to evaluate the role of PAK1 on chemosensitivity in AML by silencing PAK1 with shRNA or small molecular inhibitor. Human BMSC (HS-5) was utilized to mimic the leukemia bone marrow microenvironment (BMM) p21-activated kinase 1 high expression was shown to be associated with shorter overall survival in AML patients. The silence of PAK1 could repress cell proliferation, promote apoptosis, and enhance the sensitivity of AML cells to chemotherapeutic agents. More importantly, BMSCs induced PAK1 up-regulation in AML cells, subsequently activating the ERK1/2 signaling pathway. The effect of BMSC-mediated apoptotic-resistance could be partly reversed by knock down of PAK1.p21-activated kinase 1 is a potential prognostic predictor for AML patients. PAK1 may play a pivotal role in mediating BMM-induced drug resistance, representing a novel therapeutic target in AML. Acute myeloid leukemia (AML) is a hematological malignancy characterized by proliferation of myeloid precursors with a reduced capacity to differentiate into more mature cellular elements in the bone marrow (BM), and is correlated with genetic and epigenetic alterations. Cytogenetic and molecular genetic abnormalities are responsible for determining the response to chemotherapy and survival outcome . AlthougThe bone marrow microenvironment (BMM), which consists of soluble factors and supporting tissues, such as bone marrow stromal cells (BMSCs), extracellular matrix (ECM), provides a home for AML cells, and is responsible for disease relapse and treatment resistance . BMM plaThe p21-activated kinase (PAK) family, belonging to serine/threonine kinases, is classified into group I (PAK1-3) and group II (PAK4-6) based on structural homology and regulatory function . RecentlIn the present study, we characterized PAK1 expression in AML patients using publicly available datasets to determine its association with their clinical and molecular characteristics and clinical outcome. High expression of total and phosphorylated (active) PAK1 in the majority of AML cell lines was observed. Additionally, the inhibition of PAK1 by genetic silencing could enhance the sensitivity of AML cells to chemotherapeutic agents. Moreover, PAK1 was dramatically up-regulated in BMSC-conditioned AML cell lines, accompanied by activation of ERK1/2 signaling. Collectively, our studies support that PAK1 mediates BMM-induced drug resistance via ERK1/2 signaling pathway in AML, which provides insights for the molecular mechanism of BMM-mediated treatment resistance and targeted or combined treatment.The publicly available RNA sequencing data of 173 AML patients and complete clinical, molecular and survival data were downloaded from The Cancer Genome Atlas (TCGA) database. This database contains patients with previously untreated AML, all of them had been diagnosed, cytogenetics risk category according to the National Comprehensive Cancer Network (NCCN) guidelines. The RNA-seq and clinical data was analyzed using Gene Expression Profiling Interactive Analysis (GEPIA).Cytarabine (Ara-C), Idarubicin (IDA), and dimethyl sulfoxide (DMSO) were purchased from Actavis  and Sigma , respectively. IPA-3 was obtained from Selleck . Antibodies of PAK1 and p-PAK1 was purchased from Abcam . GAPDH, ERK1/2, p-ERK1/2, β-actin and PARP, Cleaved-PARP antibodies were obtained from Cell Signaling Technology .2 in humidified incubator. The latest authentication of cell lines was conducted by Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd. . Bone marrow primary AML samples were collected from patients during routine diagnostic assessments in Qilu Hospital, Shandong University, Jinan, China. Control samples were obtained from healthy donors. Informed consent was obtained in accordance with the Declaration of Helsinki. Mononuclear cells from bone marrow aspirates were separated by Ficoll-Paque Plus (Pharmacia LKB Biotechnology) density gradient centrifugation.Five human AML cell lines THP1, Kasumi-1, U937, HL60, and KG1, a CML cell line K562, and a T-ALL cell line Jurkat  were cultured in RPMI 1640 medium containing 10% heat-inactivated fetal calf serum with penicillin and streptomycin  at 37°C with 5% COThe shRNAs against PAK1 in a lentiviral vector with green fluorescent protein, as well as the negative control were designed and synthesized by GeneChem . High-titer lentivirus was produced in 293T cells by transfection of the lentiviral expression vector and packaging vectors GV248 using a calcium phosphate cell transfection kit according to the manufacturer’s instructions . The lentivirus was harvested 48 h later, filtered, enriched using 40% polyethylene glycol, and then used to infect AML cells. After transfection for 72 h, the efficiency was estimated by evaluation of GFP expression by fluorescence microscopy and flow cytometry. The PAK1 specific shRNA sequence used in our study was 5′-CCAAGAAAGAGCTGATTATTA-3′. We used a flow sorter to sort the transfected cells expressing GFP fluorescence for subsequent experiments.Cell viability was determined by Cell Counting Kit-8 (CCK-8). CCK-8 was obtained from BestBio . Measurements were taken 24 h after drug exposure at the indicated concentrations. Absorbance was detected at 450/630 nm by a Microplate Reader (Bio-Rad).Cell apoptosis was detected using an Annexin V-FITC/PI or Annexin V-FITC/7-AAD double stain apoptosis detection kit  according to the manufacturer’s protocols.Total RNA was extracted from cells using the TRIzol reagent . Reverse transcription was performed with an M-MLV RTase cDNA Synthesis Kit . Real-time PCR was performed with the Roche Applied Science LightCycler 480 II Real-Time PCR System using the SYBR Green gene expression assay . The following primer sets were used .PAK1: 5′-CGCCCAGAGCACACAAAATC-3′ (forward)5′-GTCCCGAGTTGGAGTGACAG-3′ (reverse)GAPDH: 5′-GCACCGTCAAGGCTGAGAAC-3′ (forward)5′-TGGTGAAGACGCCAGTGGA-3′ (reverse)5/ml overnight. Then, human AML cell lines or primary AML cells were seeded with HS-5 cells for 24 h. Cells were then treated with chemotherapy drugs or inhibitors for 24 h before performing Annexin V apoptosis assays. Co-cultured AML cells were separated from HS-5 monolayer by careful pipetting with ice-cold PBS (repeated twice). After collecting the leukemic cells, HS-5 cells monolayer was observed under microscopy (×100) to confirm that BMSC monolayer was not damaged and that <10 leukemic cells pervision field remained attached. As a control, AML cells were cultured alone and treated with chemotherapy drugs in parallel. For the transwell co-culture experiments, AML cell lines were plated inside the transwell microporous inserts (0.4 μm pore size) while underneath the inserts HS-5 cells were seeded into 12 or 6-well plates .For co-culture experiments, BMSCs (HS-5 cells) were seeded into 12 or 6-well plates at a density of 1 × 10Collected cells were lysed in lysis buffer and protease inhibitors. Fifteen to fifty micrograms of protein were separated by 10–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred onto polyvinylidene fluoride membranes. The membranes were blocked in 5% bovine serum albumin (BSA) at room temperature for 2 h, incubated with primary antibodies overnight at 4°C, and then incubated with a secondary antibody at room temperature for 1 h. Immunoreactive bands were visualized using an infrared imaging system.The cell lines which were washed with PBS were plated onto cover-slips and fixed with 4% paraformaldehyde solution for 10 min. Permeabilize cells by incubating with 0.1% Triton X-100 for 15 min. All slides were then washed with PBS and blocked with Goat serum at room temperature for 30 min. Cells were incubated with primary antibodies at 4°C overnight in a dark humidity chamber. Washed samples with PBS three times, and then incubated cells with secondary antibodies for 1 h at room temperature. In the dark, dye with DAPI for 10 min. Cells were examined immediately using Evos Fiber Illuminator  fluorescence microscope.t-test , and one-way ANOVA was used to determine the significant differences among multiple groups. Mann–Whitney U test was used for cases with unequal variances. Pearson’s Chi-square test was used to compare the clinicopathological characteristics between groups. Survival was presented with a Kaplan-Meier survival plot. Spearman correlation test was used to test the correlation between gene expressions. Differences were considered statistically significant at P < 0.05.Statistical analysis were performed using SPSS V20.0. Data were presented as mean ± standard deviation (SD) from at least three independent experiments. Statistical differences between two groups were evaluated using Student’s n = 173) and normal controls (n = 70). The results showed that the expression levels of PAK1 and PAK6 were significantly higher in AML patients than in normal controls  and control samples . PAK1 expression was significantly increased in AML patients compared to control  and relapsed/refractory AML (n = 12) than in patients with complete remission , but less t  and t  cytogenetic abnormalities . Furthermore, high PAK1 expression was shown to be associated with poorer prognosis in AML patients without FLT3, IDH1, NPMc, or RAS mutation . Specifically, patients with FLT3 mutation and high PAK1 expression had the shortest survival time, though this was not statistically different from patients with no FLT3 mutation and high PAK1 expression. COX multi-factor analysis showed that, similar to the age and cytogenetic risk status, PAK1 might be an independent prognostic factor for AML . Transfection efficiency was more than 95% evaluated by fluorescence microscope and flow fluorescence analysis . RT-qPCRTo better understand the role of PAK1 in AML, a selective non-ATP competitive PAK1 small molecule inhibitor IPA-3  was usedIt is well known that resistance to chemotherapy is the main reason for the relapse and refractory of leukemia, which resulted in fast progression of disease. Based on the fact that AML patients with PAK1 overexpression had shorter overall survival, we further explored the effect of PAK1 on drug-induced apoptosis. After PAK1 suppression by lentivirus with shRNA, THP1, and Kasumi-1 cells were treated with different concentrations of Ara-C  or IDA  for 24 h, and flow cytometry was performed to determine the apoptosis of AML cells induced by chemotherapy drugs. The results showed that knockdown of PAK1 could markedly increase Ara-C and IDA induced apoptosis of AML cells in a dose-dependent manner . Our datTHP1 cells were treated with IPA-3 (10 μM) and Ara-C (1 μM) or IDA (10 μg/L) for 24 h. The cell viability was detected by CCK-8 assay . Flow cyAccumulating evidence indicates that the interaction between hematological malignancies and BMM plays an important role in drug resistance by secreting various chemicals and contacting signals . Among tTo further elucidate whether the stroma-induced drug resistance of AML cells is due to the activation of PAK1, THP1 and Kasumi-1 cells were transfected with PAK1 shRNAs and then co-cultured with HS-5 cells. After exposure to Ara-C (2 μM) for 24 h, cell apoptosis of co-cultured cells was examined by flow cytometry. As shown in P < 0.001, We subsequently tried to figure out the mechanism of PAK1 in AML chemoresistance. Previous studies showed that PAK1 was innately associated with ERK pathway in various tumors, such as melanoma, breast cancer, malignant peripheral nerve sheath tumors and Non-small cell lung cancer . But theAcute myeloid leukemia is the most common malignant myeloid disorder in adults, and is characterized by clonal heterogeneity of hemopoietic progenitor cells. Front-line induction therapy with cytarabine and anthracycline remains a standard of care in AML . With thin vivo, and the combined treatment containing synergistic inhibition of PAK1 benefits the tumor therapy. The PAKs are a family of serine-threonine kinases that consist of six members (PAK1-PAK6), which are overexpressed in variety of cancers. PAK1 and PAK2 are overexpressed in breast and liver cancer, and promote the occurrence and development of tumors . PAK3 cuIn addition to genetic abnormalities, the BMM also exerts important influences on the chemotherapy resistance of hematological tumors. Previous reports have revealed that bone marrow stroma contributes greatly to the development of drug resistance to chemotherapy in multiple myeloma, acute, and chronic myelogenous leukemia , 2004. BIt is well known that PI3K/AKT and MEK/ERK signaling pathways play critical roles in the interaction between the BMSCs and tumor cells . PreviouIn conclusion, in the PAK family, PAK1 overexpression confers to the shorter survival and poor prognosis of AML patients. The knockdown of PAK1 could reverse chemoresistance by induction of apoptosis in AML cells. Additionally, PAK1 plays critical roles in stroma-mediated protection of AML cells, which results in BMSCs-mediated apoptosis tolerance by activating ERK1/2 signaling pathway. Our data support the strategy of targeting PAK1 to overcome chemoresistance in AML, suggesting that the combination of inhibiting PAK1 and traditional chemotherapy drugs represents a potentially novel approach. However, due to the complexity and heterogeneity of the tumor microenvironment, as well as the crosstalk among oncogenic signaling pathways, the PAK1 mediated downstream molecular mechanism need deeply investigated.Publicly available datasets were analyzed in this study. This data can be found here: The Cancer Genome Atlas (TCGA) database/Gene Expression Profiling Interactive Analysis (GEPIA).Informed consent was obtained in accordance with the Declaration of Helsinki. All laboratory experiments with primary samples were reviewed and approved by the Medical Ethics Committee of Qilu Hospital, Shandong University. The patients/participants provided their written informed consent to participate in this study.in vitro experiments. DG and QT took responsibility for statistical analyses and interpretation of data. TS and DM collected specimens and prepared the figures. CJ took responsibility for full-text evaluation and guidance, finally approval of the version to be submitted. All authors have read and approved the final manuscript.MJ and BL conceived and designed the experiments and drafted the manuscript. RJ, WeL, YZ, SD, WěL, and ML performed the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


According to the results, melatonin induced H2O2 accumulation, which was associated with the upregulation of respiratory burst oxidase homolog D (ClRBOHD) during the early response to cold stress in watermelon. Besides, melatonin and H2O2 induced the accumulation of cytoplasmic free Ca2+ ([Ca2+]cyt) in response to cold. This was associated with the upregulation of cyclic nucleotide-gated ion channel 2 (ClCNGC2) in watermelon. However, blocking of Ca2+ influx channels abolished melatonin- or H2O2-induced CBF pathway and cold tolerance. Ca2+ also induced ClRBOHD expression and H2O2 accumulation in early response to cold stress in watermelon. Inhibition of H2O2 production in watermelon by RBOH inhibitor or in Arabidopsis by AtRBOHD knockout compromised melatonin-induced [Ca2+]cyt accumulation and melatonin- or Ca2+-induced CBF pathway and cold tolerance. Overall, these findings indicate that melatonin induces RBOHD-dependent H2O2 generation in early response to cold stress. Increased H2O2 promotes [Ca2+]cyt accumulation, which in turn induces H2O2 accumulation via RBOHD, forming a reciprocal positive-regulatory loop that mediates melatonin-induced CBF pathway and subsequent cold tolerance.Cold stress is a major environmental factor that detrimentally affects plant growth and development. Melatonin has been shown to confer plant tolerance to cold stress through activating the C-REPEAT BINDING FACTOR (CBF) pathway; however, the underlying modes that enable this function remain obscure. In this study, we investigated the role of H Citrullus lanatus L.) is an economically important crop globally. Its origin can be traced to tropical and subtropical regions of Africa. Watermelon production is threatened by its susceptibility to low temperatures . After 12 h, the watermelon seedlings were exposed to chilling treatment at 4 °C for 48 h. The leaves were sampled at 3, 6, 12, and 24 h time points during chilling exposure to analyze the expression of CBF pathway genes and at 48 h for cold tolerance assay. After the initial experiments, 1 mM H2O2 was selected for the subsequent experiments.To evaluate the influences of melatonin, Helatonin , H2O2  cyt) in response to chilling stress. The protoplasts were incubated with Fluo-4 acetoxymethyl (AM) ester (a Ca2+-sensitive fluorescent dye) at 37 °C in the dark. After 30 min, the protoplasts loaded with Fluo-4/AM were treated with melatonin (10 µM), H2O2 (100 µM), or a combination of melatonin and DPI (10 µM) and then placed at 4 °C for 5 min.Protoplasts from watermelon or The maximum photosystem II quantum yield (Fv/Fm) was determined on the upper second fully expanded leaves of watermelon seedlings after 30 min of dark adaptation using a FluorCam fluorescence imaging system  . The rel2O2) content was determined as described by Willekens et al. cyt in the protoplasts was measured using Fluo-4 AM [2+]cyt accumulation levels.The cyt accumulation in watermelon protoplasts was first measured using the Fluo-4 AM ester as a fluorescent indicator of Ca2+. According to the results, chilling exposure induced the accumulation of [Ca2+]cyt in watermelon protoplasts  encodes a plasma membrane cation channel that directs extracellular Ca2+ into the cytosol [2+]cyt accumulation, both melatonin and H2O2 promoted the cold-induced ClCNGC2 upregulation; however, the effect was blocked by DPI pretreatment. These results demonstrate that H2O2 participates in melatonin-induced [Ca2+]cyt accumulation under chilling stress.To investigate whether Catoplasts . Notably cytosol ,49. In l3 is an inhibitor of Ca2+ influx channels cyt accumulation under cold stress, accompanied by ClCNGC2 upregulation  ,63,64. VR) genes ,65. Seven plants ,29,66. Hgulation . Arabido cytosol ,49. Heretermelon . These r2O2 and Ca2+ signal have been well documented in various physiological actions, especially in defense against abiotic stressors cyt accumulation, which boosts H2O2 accumulation by triggering the ClRBOHD expression, forming a reciprocal positive-regulatory loop that induces the expression of CBF pathway genes and subsequent cold tolerance. This is the first study to investigate the interplay between H2O2 and Ca2+ signaling in melatonin-mediated cold tolerance to the best of our knowledge. However, other components of melatonin signaling in plant response to cold stress need to be further explored in the future.At present, the signaling mechanisms underlying melatonin-induced cold tolerance in watermelon are still elusive. This study reveals an intricate signaling cascade of melatonin-induced cold stress tolerance in watermelon. Melatonin induces H


Mechanisms for the protective effects of the antioxidant enzyme peroxiredoxin-6 (Prx-6) on hippocampal cells during oxygen-glucose deprivation/reoxygenation (OGD/R) were investigated. Using the methods of fluorescence microscopy, inhibitory analysis, vitality tests and PCR, it was shown that 24-h incubation of mixed hippocampal cell cultures with Prx-6 does not affect the generation of a reversible phase of a OGD-induced rise in Ca2+ ions in cytosol ([Ca2+]i), but inhibits a global increase in [Ca2+]i in astrocytes completely and in neurons by 70%. In addition, after 40 min of OGD, cell necrosis is suppressed, especially in the astrocyte population. This effect is associated with the complex action of Prx-6 on neuroglial networks. As an antioxidant, Prx-6 has a more pronounced and astrocyte-directed effect, compared to the exogenous antioxidant vitamin E (Vit E). Prx-6 inhibits ROS production in mitochondria by increasing the antioxidant capacity of cells and altering the expression of genes encoding redox status proteins. Due to the close bond between [Ca2+]i and intracellular ROS, this effect of Prx-6 is one of its protective mechanisms. Moreover, Prx-6 effectively suppresses not only necrosis, but also apoptosis during OGD and reoxygenation. Incubation with Prx-6 leads to activation of the basic expression of genes encoding protective kinases—PI3K, CaMKII, PKC, anti-apoptotic proteins—Stat3 and Bcl-2, while inhibiting the expression of signaling kinases and factors involved in apoptosis activation—Ikk, Src, NF-κb, Caspase-3, p53, Fas, etc. This effect on the basic expression of the genome leads to the cell preconditions, which is expressed in the inhibition of caspase-3 during OGD/reoxygenation. A significant effect of Prx-6 is directed on suppression of the level of pro-inflammatory cytokine IL-1β and factor TNFα, as well as genes encoding NMDA- and kainate receptor subunits, which was established for the first time for this antioxidant enzyme. The protective effect of Prx-6 is due to its antioxidant properties, since mutant Prx-6  leads to polar opposite effects, contributing to oxidative stress, activation of apoptosis and cell death through receptor action on TLR4.Ischemia-like conditions reflect almost the entire spectrum of events that occur during cerebral ischemia, including the induction of oxidative stress, Ca The protective effect of Prx-6 is more pronounced in astrocytes, where there is a complete suppression of the OGD-induced increase in [Ca2+]i, whereas in neurons—there is 70% suppression of this phase of the OGD-induced signal. The protective effect of Prx-6 is provided by endocytosis during 24-h incubation of cell cultures.Therefore, the antioxidant enzyme Prx-6 is able to protect the brain cells from necrotic death during ischemia (OGD) by inhibiting the phase of the global increase in i and NO production, we were able to show that in response to OGD, there is an increase in NO production in neurons  production can contribute significantly to the toxic effects of OGD. By simultaneously measuring the dynamics of i, decreases the percentage of the dying cells, promoting suppression of apoptosis under OGD conditions and during reoxygenation via changing baseline expression of the genes encoding proteins which exert a harmful effect of genes encoding protective proteins. The possibility of suppressing Ca2+-signaling of the neurons and astrocytes during ischemia or hypoxia by antioxidants taxifolin and Vit E has been shown in our previous studies. At the same time, the role of the antioxidant enzyme Prx-6 in regulation of Ca2+-dynamics in neurons and astrocytes is shown for the first time. A significant decrease in expression of genes encoding AMPAR and KAR may contribute significantly to the suppression of OGD-induced Ca2+-signals in neurons and especially to the global increase in [Ca2+]i. The effect of Prx-6 is multiple: it protects both neurons and astrocytes against OGD-induced neurotoxic factors, but its protective effect is most pronounced for astrocytes. This selectivity for astrocytes may be explained by the increased level of Prx-6 expression especially in astrocytes and, hence, by their better adaptation to the protective effect of exogenous additives of Prx-6. Cell-type-specific expression patterns are shown for Prxs isoforms, while Prx-1 and Prx-6 are predominantly expressed in astrocytes and oligodendrocytes and Prx-2-5—in neurons i is the leading intracellular mediator in these processes. An increase in its level contributes to enhancement of ROS production in mitochondria, and its global increase leads to necrotic death of the brain cells during ischemia [Mitochondria are generators and targets for intracellular ROS at one time. Mitochondrial dysfunctions induced by oxidative stress during ischemia can lead to delayed death of the brain cells . [Ca2+]iischemia . Suppres2O2 can freely penetrate into the cytosol, performing signaling functions, and its excessive concentrations are normally converted by antioxidant enzymes, including peroxiredoxins i, hippocampal cell cultures were loaded with Fura-2 . The cells were stained with the probe dissolved in Hank‘s balanced salt solution (HBSS) composed of (mM): 156 NaCl, 3 KCl, 2 MgSO4, 1.25 KH2PO4, 2 CaCl2, 10 glucose, and 10 HEPES, pH 7.4. To measure [Ca2+]i, we used the system based on an inverted motorized microscope Leica DMI6000B with a high-speed monochrome CCD-camera HAMAMATSU C9100. For excitation and registration of Fura-2 fluorescence, we used FU-2 filter set  with excitation filters BP340/30 and BP387/15, beam splitter FT-410, and emission filter BP510/84. Illuminator Leica EL6000 with a high-pressure mercury lamp was used as a source of excitation light. To distinguish neurons and astrocytes, we used short-term applications of 35 mM KCl and 10 µM ATP before the main experiments. This method was described in detail in our previous work i and necrosis.Thus, exogenous Prx-6 has a complex protective effect on hippocampal cells during ischemia with tendency towards more selective protection of astrocytes. Incubation with Prx-6 changes the basic antioxidant status of cells through changes in the expression of key genes, resulting in the suppression of ROS production by mitochondria under OGD conditions. The anti-apoptotic effect of Prx-6 also occurs through an increase in the expression of genes encoding protective kinases and transcription factors while suppressing proteins with toxic effects. The protective effect of Prx-6 also includes its anti-inflammatory effect and suppression of the expression of glutamate receptors involved in the excitation of cells, thereby contributing to inhibition of global increase in [Ca