annexin-V Binding Research Articles

Theaflavins Induce Autophagy in Ehrlich’s Ascites Carcinoma Cells both In vivo and In vitro

Aims: To investigate the efficacy of Theaflavins to induce autophagy and its tumoricidal activity towards Ehrlich’s Ascites Carcinoma cells. Background: The apoptosis-inducing role of Theaflavins against cancer is reported. Autophagy, a cellular mechanism under stress, occurs either as a survival process or Type-II programmed-cell death in the presence/absence of apoptosis. The report of Theaflavins inducing autophagy against cancer is poor. Objective: Here, for the first time, the investigation for the anti-tumor efficacy of Theaflavins via autophagy in EAC was attempted. Methods: EAC-bearing mice were treated orally with Theaflavins (10 mg/kg b.w.) every alternate day with a total of 27 doses. Body weight, tumor volume and survivability were recorded. Tumoricidal and cellular dehydrogenase activity, in vivo and in vitro, were studied using Trypan-blue exclusion and MTT assay respectively. Theaflavins-treated EAC cells were subjected to Monodansylcadaverine- staining. LC3II turnover and LC3I conversion were detected by western blotting. Apoptosis up to 12 h TF-treatment was estimated by AnnexinV binding. Results: This is the first report of Theaflavins inducing autophagy in EAC cells in vivo and in vitro. Oral Theaflavins treatment restricted excessive body-weight increase due to tumors, reduced tumor volume, and increased survivability of tumor-bearing mice. Theaflavins caused EAC cell death (~8% in vitro, ~30% in vivo), significantly reduced metabolic activity, and created conspicuous vacuolization in surviving cells. Resultant vacuoles (in vitro, 6 h) were marked as autophagosomes by Monodansylcadaverine-staining. Autophagy was confirmed by LC3II augmentation. No significant apoptosis was observed up to 12 h TF-treatment in vitro. Conclusion: Theaflavins were efficient inducing autophagy and Type-II PCD in EAC cells. Notably, Theaflavins induced autophagy prior to apoptosis in vitro.

Cell Death in Leishmania donovani promastigotes in response to Mammalian Aurora Kinase B Inhibitor– Hesperadin

Deciphering how hesperadin, a repurposed mammalian aurora kinase B inhibitor, affects the cellular pathways in Leishmania donovani might be beneficial. This investigation sought to assess the physiological effects of hesperadin on promastigotes of L. donovani, by altering the duration of treatment following exposure to hesperadin. Groups pre-treated with inhibitors such as EGTA, NAC, and z-VAD-fmk before hesperadin exposure were also included. Morphological changes by microscopy, ATP and ROS changes by luminometry; DNA degradation using agarose gel electrophoresis and metacaspase levels through RT-PCR were assessed. Flow cytometry was used to study mitochondrial depolarization using JC-1 and MitoTracker Red; mitochondrial-superoxide accumulation using MitoSOX; plasma membrane modifications using Annexin-V and propidium iodide, and lastly, caspase activation using ApoStat. Significant alterations in promastigote morphology were noted. Caspase activity and mitochondrial-superoxide rose early after exposure whereas mitochondrial membrane potential demonstrated uncharacteristic variations, with significant functional disturbances such as leakage of superoxide radicals after prolonged treatments. ATP depletion and ROS accumulation demonstrated inverse patterns, genomic DNA showed fragmentation and plasma membrane showed Annexin-V binding, soon followed by propidium iodide uptake. Multilobed macronuclei and micronuclei accumulated in hesperadin exposed cells before they disintegrated into necrotic debris. The pathologic alterations were unlike the intrinsic or extrinsic pathways of classical apoptosis and suggest a caspase-mediated cell death most akin to mitotic-catastrophe. Most likely, a G2/M transition block caused accumulation of death signals, disorganized spindles and mechanical stresses, causing changes in morphology, organellar functions and ultimately promastigote death. Thus, death was a consequence of mitotic-arrest followed by ablation of kinetoplast functions, often implicated in L. donovani killing.

Development of chromone-thiazolidine-2,4-dione Knoevenagel conjugates as apoptosis inducing agents

Overexpression of Bcl-2 protein is a predominant hallmark of disturbed apoptotic pathway in most of the cancers. Herein, chromone-linked thiazolidinediones were designed and synthesized to target Bcl-2 for regulating anti-apoptotic proteins. The study on in vitro cancer cell lines revealed the presence of compounds 8a, 8k, 8l, and 8n, which were found to have good to moderate anti-proliferative activity (with an IC50 concentration less than 10 µM). Among them, 8l depicted the highest cytotoxicity on the A549 cell line with an IC50 of 6.1 ± 0.02 µM. Aberrantly, the compounds displayed less toxicity towards human embryonic kidney HEK cells underlining its selectivity. The DCFDA study revealed a gradual increase in the ROS generation of 8l, followed by its quantification by flow analysis. Similarly, the studies including DAPI, AO/EtBr and Annexin-V binding clearly elucidated the DNA damage, membrane integrity prospects, and insights for early and late apoptotic phases. Markedly, the Bcl-2-FITC anti-body study revealed that compound 8l reduced the expression of anti-apoptotic proteins by 79.1 % compared to the control at 9 µM concentration. In addition, the molecular docking study provided the impending scope of these hybrids, showing promising interaction with the Mcl-1 target (member of the Bcl-2 family) with comparable binding affinities.

Exploring etofenamate hydrazide-hydrazone/copper(II) complexes: Synthesis, anticancer activity, carbonic anhydrase IX inhibition and docking studies

Hydrazide-hydrazone derivatives have garnered significant interest from researchers globally due to their wide range of biological activities, including antiviral, anticancer, and anti-inflammatory properties. In this study, a novel series of etofenamate hydrazide-hydrazone compounds (2a-2s) and their Cu(II) complexes (3a-3s) were designed and synthesized. The compounds were characterized using various analytical techniques such as FT-IR, 1H NMR, 13C NMR, MS, and elemental analysis. Additionally, the compound 2c and Cu(II) hydrazone complex 3a were further characterized using single X-ray crystallography. The anti-proliferative activity of the compounds was evaluated against Ishikawa human endometrial cancer cell line and non-tumour L929 cells using MTT assay. Additionally, the apoptotic potential of the compounds was investigated through caspase-3 activity, Bax and Bcl-2 gene expression analysis, and annexin-V binding. Furthermore, carbonic anhydrase IX activity and in silico studies were conducted to elucidate the mechanism of action. Overall, compound 3s demonstrated significant antiproliferative effects with an IC50 value of 0.27±0.01 µM against Ishikawa cells.

MK591 (Quiflapon), a 5-lipoxygenase inhibitor, kills pancreatic cancer cells via downregulation of protein kinase C-epsilon.

Pancreatic tumors and cell lines derived from them exhibit elevated expression of 5-lipoxygenase (5-Lox), whereas non-tumor glands or normal cells do not exhibit this overexpression. Arachidonic acid stimulates pancreatic cancer cell growth via metabolic conversion through the 5-Lox pathway, and inhibition of 5-Lox activity decreases the viability of pancreatic cancer cells. However, the downstream signaling mechanisms through which 5-Lox exerts its effects on the survival of pancreatic cancer cells remain to be elucidated. The effects of 5-Lox inhibition on cell proliferation, apoptosis, and invasive potential were investigated in pancreatic cancer cells. The protein expression was analyzed by Western blot. Apoptosis was analyzed by Annexin-V binding assay and by detecting the degradation of chromatin-DNA to nucleosomal fragments. The protein kinase C-epsilon (PKCε) activity was measured by an immunoprecipitation-kinase assay. The in vivo effects of MK591 were evaluated in pancreatic tumor xenograft model. MK591, a specific inhibitor of 5-Lox activity, killed pancreatic cancer cells via induction of apoptosis, involving externalization of phosphatidylserine, cleavage of PARP (poly-ADP ribose polymerase) and degradation of chromatin DNA to nucleosomes. MK591 effectively blocked in vitro invasion and soft-agar colony formation by pancreatic cancer cells and decreased pancreatic tumor growth in nude mice xenografts. Furthermore, inhibition of 5-Lox downregulated K-Ras and inhibited phosphorylation of c-Raf and ERKs. Interestingly, 5-Lox inhibition induced apoptosis in pancreatic cancer cells without the inhibition of Akt but the protein level of PKCε was dramatically downregulated. Furthermore, inhibition of 5-Lox decreased the phosphorylation of Stat3 at Serine-727. Pre-treatment of pancreatic cancer cells with peptide activators of PKCε prevented apoptosis induced by 5-Lox inhibition, suggesting that the mechanism by which 5-Lox inhibition causes cell death in pancreatic cancer involves downregulation of PKCε. The combination of low doses of MK591 and gemcitabine synergistically reduced the oncogenic phenotype and killed pancreatic cancer cells by inducing apoptosis. These findings indicate that inhibition of 5-Lox interrupts an Akt-independent, PKCε-dependent survival mechanism in pancreatic cancer cells and suggest that metabolism of arachidonic acid through the 5-Lox pathway plays an integral part in the survival of pancreatic cancer cells via signaling through PKCε, an oncogenic, pro-survival serine/threonine kinase.

Evaluation of Synergistic Anti-Leukemic Efficacy of PHI-101 in Preclinical Model of FLT3-ITD Acute Myeloid Leukemia

Background: AML is a heterogeneous disease with an overall cure rate of 40-50%. Some types of AML have even poorer prognosis with FLT3 mutant AML patients having only a 20-25% cure rate with chemotherapy alone. In order to improve the outcome for this high-risk AML, a number of tyrosine kinase inhibitors (TKI) have been developed and to date. Given the difficulty of identifying appropriate targets and treatment options for AML, combining drug options for AML treatment may produce clinical benefits. PHI-101 is a selective next-generation type I FLT3 inhibitor that shows potent anti-leukemic activity against AML samples harboring FLT3-ITD and/or TKD mutations. In a clinical trial, PHI-101 demonstrates significant clinical responses and tolerability in refractory or relapsed AML patients as monotherapy. To investigate the optimal way to combine PHI-101 with other therapies, a number of other drugs utilized in treating AML were tested both, in vitro and in vivo to determine synergistic or additive effects that might improve efficacy for relapsed/refractory/elderly FLT3 mutant AML patients. Study Design and Methods: Combination effects of simultaneous versus sequential treatment of PHI-101 with other agents, including daunorubicin, cytarabine, venetoclax (Ven), and azacytidine (Aza) were tested using human leukemia cell lines harboring FLT3-ITD mutations (MV4-11, Molm13, Molm14) or FLT3-wild type as controls. The cell growth inhibition was assessed by CCK-8, MTT and/or CellTiter-Glo assays as well as annexin V-binding by flow cytometry and the Chou-Talalay method was used to calculate the combination index (CI. using Compusyn software. The potential mechanisms of effect on combination therapy were explored by Western blotting of a variety of signal transduction pathways including FLT3 phosphorylation and downstream pathways BCL-2 family expression, and DNA damage. The MV4-11 xenograft mouse model was used to assess in vivo efficacy of PHI-101 combination therapy. Results: Among the substances tested for combination therapy with PHI-101 in FLT3-ITD cell lines MV4-11 and Molm14 cell lines, Aza and Ven treatment both showed synergy with both simultaneous and sequential treatment. Ven was highly synergistic when used simultaneously with PHI-101 in MV4-11 cells. Cell growth inhibition effect (GI 50) of PHI-101, Aza, and Ven used alone was measured first to calculate the CI in combination with increasing concentrations of each drug together of treatment for 72hrs. The GI 50 of PHI-101 in FLT3-mutant cell lines (MV4-11, Molm13, Molm14) were 7.0, 3.8, and 3.4 nM, respectively. The GI 50 of the gilteritinib was 5.7, 10.2, and 10.8 nM, respectively. On the other hand, the GI 50 of Aza were 198.9, 140.4, and 85.4 nM, that of Ven were 38.2, 8.4, and 56.5 nM. PHI-101 was the most efficient antitumor agent compared to other drugs tested in this study. The CI of Aza and PHI-101 was 0.01216 (EC 50), 0.05981 (EC 75), 0.29468 (EC 90) in MV4-11 cells. The observed synergistic effects were confirmed in the other FLT3-ITD cell lines as well. PHI-101 and venetoclax combined therapy in MV4-11 showed synergism (CI<0.1), and the CI was 0.00002 (EC 50), 0.00104 (EC 75), 0.01243 (EC 90). By Western blotting, the combination of Aza or Ven with PHI-101 resulted in a significant induction of p-H2AX and PARP cleavage in the FLT3-ITD cell lines (MV4-11), but not in FLT3-WT cell lines (THP-1). To further validate the synergistic anti-leukemic activity of PHI-101 with these agents, MV4-11-derived mouse xenograft models were generated and treated at various doses. In the MV4-11-xenograft model, PHI-101 alone or combined with Ven or Aza significantly decreased tumor volume. In line with the cell viability data, Ven and Aza combined with PHI-101 induced significant cell-killing in vivo. Conclusion: Treatment of AML cells with the combination of Ven or Aza with PHI-101 in vitro induced rapid induction of apoptosis and inhibition of cell growth that showed significant synergy. In vivo data showed improved efficacy for the combination treatment with these agents. In vitro and in vivo data from the preclinical AML models provides a rationale to evaluate the activity of PHI-101 in combination for patients who are ineligible for more intensive chemotherapeutic induction therapy. Improved anti-leukemic efficacy by combined therapy with PHI-101 represent a promising therapeutic strategy for newly diagnosed or R/R FLT3-mutant AML patients

Enriched Signalling Pathways in Venetoclax-Relapsed Chronic Lymphocytic Leukemia (CLL) Cells and Targeting Using a Protac-Based Bcl-2/Bcl-Xl Degrader

Venetoclax is a specific inhibitor of Bcl-2, the key protein which protects CLL cells from intrinsic apoptosis, whereas the Bruton's Tyrosine Kinase (BTK) inhibitor ibrutinib kills CLL cells via blockade of B-cell receptor (BCR) signalling. Unfortunately, some patients develop treatment resistance after receiving these agents in combination or sequentially. We aimed to evaluate whether CLL cells collected upon venetoclax and ibrutinib relapse retain survival dependency on alternative targets of the BCR pathway and Bcl-2 family, and to propose new vulnerabilities based on gene expression changes. Using single cell RNA sequencing (sc-RNA-seq), we analyzed paired samples from a CLL patient collected at baseline and at relapse, which occurred after cessation of fixed-duration treatment with venetoclax and ibrutinib. The proportion of cells in each CLL cluster was vastly altered at relapse. Cluster 3 represented 4% of CLL cells at baseline versus 36% at relapse and had a high BCR score, including high expression of MAP3K8 FOSB8 and NFKB1 genes. Moreover, at relapse, it contained more CLL cells expressing transcripts from the Bcl-2 family of anti-apoptotic proteins, including Bcl-2 and Bcl-xL. We also integrated sc-RNA-seq data from three primary CLL samples collected at relapse after venetoclax and ibrutinib treatment. The four clusters of CLL cells identified were enriched for BCR, interleukin-mediated, ErbB and the Fc epsilon receptor I signalling pathways. Importantly, most of the relapsed CLL cells which expressed high levels of Mcl-1 or Bcl-xL also expressed Bcl-2, suggesting that these resistant CLL cells became dependent on various anti-apoptotic proteins for survival. These observations led us to test Bcl-2/Bcl-xL targeting PROTACs PZ18753b and WH25244 in venetoclax-resistant CLL. These PROTACs were derived from navitoclax (Bcl-2/Bcl-xL dual inhibitor) linked to a VHL E3 ligase ligand to target Bcl-2 and Bcl-xL for degradation, with improved specificity to cancer cells while sparing platelets. We used three models of venetoclax-resistant CLL: (1) OSU-CLL cells expressing mutant Bcl-2 (G101V, F104L, R107-110dup or A113G), (2) treatment-naïve primary CLL cells co-cultured with NK.Tert stromal cells for 24h (CLL+stroma), and (3) primary CLL cells collected at venetoclax relapse. CLL cells were treated with venetoclax, navitoclax, PZ18753b, WH25244, or negative controls (NC) lacking VHL recruitment, PZ18753b-NC or WH25244-NC. Protein degradation was evaluated by Western Blot; viability, by Cell Titer Glo; and apoptosis, by flow cytometric detection of active BAX and BAK, mitochondrial cytochrome C, and AnnexinV binding. WH25244 was superior to venetoclax at targeting OSU-CLL cells expressing either wildtype or mutant Bcl-2, upon 72h treatment. Cell death by WH25244 was preceded by degradation of Bcl-xL and partial degradation of Bcl-2, despite presence of Bcl-2 mutations. In treatment-naïve primary CLL cells, Bcl-xL degradation by PZ18753b and WH25244 proceeded in a dose-, time- and VHL-dependent manner. The potency of these PROTACs fell in between that of venetoclax and navitoclax, with mitochondrial apoptosis occurring at low nanomolar concentrations known to spare human platelets. Stepwise, activation of BAK and BAX, and cytochrome C release were evident at 14h of treatment, followed by phosphatidyl serine exposure at 18h of treatment. A rightward shift in the dose-response curves of PZ18753b (16-fold) and WH25244 (8-fold) was noted for CLL+stroma cells relative to paired CLL cells treated in suspension. This reduction in potency was associated with (1) uptake of the PROTACs by NK.Tert cells, since Bcl-xL degradation was noted in stromal cells yet without evident toxicity, and (2) up to 2.5-fold increased levels of Bcl-xL in CLL+stroma, which shifted the dose-dependent degradation of Bcl-xL. Finally, 2 out of 3 venetoclax-relapsed primary CLL samples showed more active BAK and cytochrome C release upon treatment with WH25244 than venetoclax, evaluated at 10 nM. In conclusion, WH25244 is a PROTAC-based Bcl-2/Bcl-xL degrader with the potential to overcome venetoclax-resistant CLL dependent on Bcl-xL and mutant Bcl-2. Relative to its precursor, navitoclax, it shows increased potency against CLL cells and decreased toxicity against platelets in vitro, due to its VHL-dependent activity and minimal expression of VHL in platelets.

FT538, iPSC-Derived NK Cells Are Potent Inducers of Apoptosis in AML Cells and Their Effect Is Synergistic in Combination with Approved Therapeutic Strategies

Background: Natural killer (NK) cell-based therapies have been increasingly studied with their advantages of less treatment-related toxicity and lack of HLA restriction. Induced pluripotent stem cell (iPSC)-derived NK cells offer an opportunity for a standardized, off-the-shelf treatment with the potential to treat a wider patient population. FT538 cells are triple gene-edited iPSC-derived NKs that express a high affinity non-cleavable CD16 maintaining constant activity, a CD38 knock out preventing CD38 antibody-induced fratricide, and an IL-15/IL-15R fusion preventing the need for cytokine administration, the main source of side effects in NK cell-based therapies. Several studies have shown success in treating AML with NK cell therapy, but the effect of iPSC-derived NK cell therapy in AML is unexplored. We hypothesized that FT538 cells may be able to target high-risk AML that is traditionally resistant to most chemotherapeutic agents and synergize with Venetoclax, 5-azacytidine, Cytarabine, and Gilteritinib. Methods: To examine the effect of the FT538 cells on AML cells, we co-cultured FT538 cells at ratios of 2:1, 4:1, and 8:1 with 8 AML cell lines: OCI-AML2, OCI-AML3, THP1, U937, Molm-13, Molm-14, MV4-11, and Kasumi-1, and with cells from 11 different AML patients. We analyzed the NK cell-induced apoptosis through an Annexin-V binding assay using IncuCyte live cell imaging. As Molm-13 and MV4-11 cell lines are FLT3 mutated, to determine the combination effect of FLT3 inhibitors with FT538 cells, we treated Molm-13 and MV4-11 cells with Gilteritinib for 24 hours before seeding the FT538 cells and co-culturing overnight. The effects of the combination were then analyzed through flow cytometry with a Beckman Coulter CytoFlex. Finally, to determine the combinatorial effect of the FT538 cells with approved therapies, we treated OCI-AML3 and U937 cell lines with Venetoclax, Azacitidine, and Cytarabine. After treatment, we performed flow cytometry to measure the leukemic cell death following the combinatorial therapies. Results: We observed FT538 NK-mediated killing in all the leukemic cell lines tested. A two-way ANOVA determined that an increase in the FT538 to AML cell ratio resulted in an increase in NK cell-mediated killing of AML cells (p<0.0001). Interestingly, the number of apoptotic AML cells was 1.5 to 6.1-fold higher in the OCI-AML2, OCI-AML3, U937, and THP1 cell lines than in the Molm-13, Molm-14, MV4-11, and Kasumi-1 cell lines (p<0.0001). This could be related to the FLT3 mutations in the Molm-13, Molm-14, and MV4-11 cell lines or to the TP53 mutation in the Kasumi-1 cell line. Further, to validate this finding in primary AML cells, we observed a ratio-dependent effect on FT538-mediated killing of AML cells. However, we did not observe any statistical difference by mutation, though this could be related to the limited sample size for specific mutations. Finally, flow cytometric analysis of the effects of combination treatment demonstrated that the FT538 cells synergize with Venetoclax, or Cytarabine, or Azacitidine, or Gilteritinib (p<0.05) in the killing of AML cells. This could be because NKG2DL upregulation has been shown in AML cells treated with Venetoclax and FLT3 inhibitors and in Cytarabine-resistant AML cells, and the NKG2D/NKG2DL pathway is a main mechanism of NK cell-mediated killing. Conclusion: iPSC-derived NK therapy offers a standardized, off-the-shelf option for NK cell therapies. FT538 iPSC-derived NK cells induce apoptosis in AML cell lines and patient samples in a dose-dependent manner and show a synergistic effect with Venetoclax, Gilteritinib, Azacitidine, and Cytarabine. Therefore, iPSC-derived NK cell therapy may be a promising possibility for AML treatment, particularly for patients resistant to standard therapies.

PD-L1 Is Involved in the Development of Non-Hodgkin's Lymphoma by Mediating Circulating Lymphocyte Apoptosis.

Lymphocyte apoptosis plays a crucial role in tumor-induced immunosuppression. Programmed death ligand-1 (PD-L1) blocks lymphocyte activation via its receptor, PD-1. However, PD-L1/PD-1 expression and its role in enhancing immune suppression in non-Hodgkin lymphoma (NHL) have not been identified. The purpose of the study was to assess PD-L1/PD-1 expression in circulating lymphocytes in NHL and its role in immunosuppression. Twenty newly diagnosed NHL patients and twenty normal volunteers were enrolled in the study. PD-L1/PD-1 expression in circulating lymphocytes and the apoptosis of lymphocyte subsets were assessed using flow cytometry. The findings revealed that the PD-L1 expression in circulating CD3+, CD3+CD4+, CD3+CD8+, and CD20+ lymphocytes were dramatically upregulated in NHL patients (p < 0.001), whereas peripheral lymphocytes expressed low levels of PD-1. Compared with normal volunteers, a significant increase in lymphocyte apoptosis was revealed by annexin-V binding on T and B lymphocytes (p < 0.001). Peripheral lymphocytes expressing PD-L1 were four times more vulnerable to apoptosis than those expressing PD-1. Our findings imply that PD-L1 upregulation contributes to NHL development by promoting circulating lymphocyte apoptosis. This research adds to our understanding of the function of the PD-L1/PD-1 pathway in tumor evasion, establishing a novel therapeutic target in NHL. The results offer additional evidence for the immunomodulatory role of PD-L1 in circulating lymphocytes, providing a rationale for further investigations into immunological dysfunctions resulting from NHL. PD-L1+ lymphocytes could be employed as a biomarker to assess the effectiveness of immune systems and predict illness in patients with NHL.

Cytotoxic Activity of Melatonin Alone and in Combination with Doxorubicin and/or Dexamethasone on Diffuse Large B-Cell Lymphoma Cells in In Vitro Conditions.

Melatonin (MLT), a pineal gland hormone, not only regulates circadian and seasonal rhythms, but also plays an important role in many aspects of human physiology and pathophysiology. MLT is of great interest as a natural substance with anti-cancer activities. The aim of this study was to assess the cytotoxicity and apoptosis of MLT, used alone or in combination with one of the most active anti-cancer drugs, doxorubicin (DOX), and a well-known anti-inflammatory drug, dexamethasone (DEX), on a diffuse large B-cell lymphoma (DLBCL)-derived cell line. The cytotoxicity and cell cycle distribution were measured using propidium iodide staining, while apoptosis was assessed using the annexin-V binding method. Additionally, to elucidate the mechanisms of action, caspase-3, -8, and -9 and a decline in the mitochondrial potential were determined using flow cytometry. MLT inhibited cell viability as well as induced apoptosis and cell cycle arrest at the G0/G1 phase. The pro-apoptotic effect was exerted through both the mitochondrial and caspase-dependent pathways. Furthermore, we observed increased cytotoxic and pro-apoptotic activity as well as the modulation of the cell cycle after the combination of MLT with DOX, DEX, or a combination of DOX + DEX, compared with both drugs or MLT used alone. Our findings confirm that MLT is a promising in vitro anti-tumour agent that requires further evaluation when used with other drugs active against DLBCL.

Impaired Platelet Function and Thrombus Formation in PDE5A-Deficient Mice.

Intracellular cyclic GMP (cGMP) inhibits platelet function. Platelet cGMP levels are controlled by phosphodiesterase 5A (PDE5A)-mediated degradation. However, the exact role of PDE5A in platelet function and thrombus formation remains poorly understood. In this study, we characterized the role of PDE5A in platelet activation and function. Platelets were isolated from wild type or PDE5A-/- mice to measure platelet aggregation, activation, phosphatidylserine exposure (annexin-V binding), reactive oxygen species (ROS) generation, platelet spreading as well as clot retraction. Cytosolic calcium mobilization was measured using Fluo-4 AM by a microplate reader. Western blot was used to measure the phosphorylation of VASP, ERK1/2, p38, JNK, and AKT. FeCl3-induced arterial thrombosis and venous thrombosis were assessed to evaluate the in vivo hemostatic function and thrombus formation. Additionally, in vitro thrombus formation was assessed in a microfluidic whole-blood perfusion assay. PDE5A-deficient mice presented significantly prolonged tail bleeding time and delayed arterial and venous thrombus formation. PDE5A deficiency significantly inhibited platelet aggregation, ATP release, P-selectin expression, and integrin aIIbb3 activation. In addition, an impaired spreading on collagen or fibrinogen and clot retraction was observed in PDE5A-deficient platelets. Moreover, PDE5A deficiency reduced phosphatidylserine exposure, calcium mobilization, ROS production, and increased intracellular cGMP level along with elevated VASP phosphorylation and reduced phosphorylation of ERK1/2, p38, JNK, and AKT. In conclusion, PDE5A modulates platelet activation and function and thrombus formation, indicating that therapeutically targeting it might be beneficial for the treatment of thrombotic diseases.

Effect of platelet exosomes loaded with doxorubicin as a targeted therapy on triple-negative breast cancer cells.

Exosomes have come to the fore in drug delivery systems due to their biological-based and immune-suppressing properties. In this study, we investigated the effect of doxorubicin loading of exosomes isolated from human platelets on breast cancer.Exosomes released from ADP (1µM)-activated platelets were isolated by the ultracentrifugation method, and their size and charge were measured with a TEM and zeta sizer. Then doxorubicin (Dox) loading into exosomes (PLT-Exo-Dox) was done by electroporation and incubated with MDA-MB-231 cells. In exosome characterization, CD62 positivity was higher in platelet pellets, while CD9 positivity was higher in released exosomes. The size of PLT-Exo and PLT-Exo-Dox was 82.02 ± 5.21nm and 116 ± 3.73nm, with a polydispersity index of 0.26 ± 0.04 and 0.39 ± 0.06, and the Zeta potential was -16.45mV and 24.07mV, respectively. The encapsulation efficiency of the preparation was 86.02 ± 6.16%, with a drug loading capacity of 4.75 ± 0.16µg/µg of the exosome. In MDA-MB-231 cells, PLT-Exo increased cell viability, while PLT-Exo-Dox decreased in 24h. The Annexin-V binding level and Bax gene expression were increased in PLT-Exo-Dox and Bcl-2 gene expression was decreased. This study will shed light on the development of release systems that can be effective with chemotherapeutic agents by using exosomes released by cells in the development of personalized treatments.

Hypercoagulability and Inflammatory Markers in a Case of Congenital Thrombotic Thrombocytopenic Purpura Complicated by Fetal Demise

Congenital thrombotic thrombocytopenic purpura (cTTP) is a rare disorder caused by an inherited genetic deficiency of ADAMTS13 and affects less than one per million individuals. Patients who are diagnosed with TTP during pregnancy are at increased risk of maternal and fetal complications including fetal demise. We present a case of a 32-year-old G3P0 (gravida 3, para 0) who presented at 20 weeks gestation with a new diagnosis of congenital TTP (cTTP) and fetal demise. We describe the pathophysiology of pregnancy complications in a patient with cTTP using platelet procoagulant membrane dynamics analysis and quantitative proteomic studies, compared to four pregnant patients with gestational hypertension, four pregnant patients with preeclampsia, and four healthy pregnant controls. The cTTP patient had increased P-selectin, tissue factor expression, annexin-V binding on platelets and neutrophils, and localized thrombin generation, suggestive of hypercoagulability. Among 15 proteins that were upregulated, S100A8 and S100A9 were distinctly overexpressed. There is platelet-neutrophil activation and interaction, platelet hypercoagulability, and proinflammation in our case of cTTP with fetal demise.

Effect of Mercury on Membrane Proteins, Anionic Transport and Cell Morphology in Human Erythrocytes.

Mercury (Hg) is a heavy metal widespread in all environmental compartments as one of the most hazardous pollutants. Human exposure to this natural element is detrimental for several cellular types including erythrocytes (RBC) that accumulate Hg mainly bound to the SH groups of different cellular components, including protein cysteine residues. The cellular membrane represents a major target of Hg-induced damage in RBC with loss of physiological phospholipid asymmetry, due to phosphatidylserine (PS) exposure to the external membrane leaflet. To investigate Hg-induced cytotoxicity at the molecular level, the possible interaction of this heavy metal with RBC membrane proteins was investigated. Furthermore, Hg-induced alterations in band 3 protein (B3p) transport function, PS-exposing macrovesicle (MVs) formation and morphological changes were assessed. For this aim, human RBC were treated in vitro with different HgCl2 concentrations (range 10-40 µM) and the electrophoretic profile of membrane proteins as well as the expression levels of Ankyrin and Flottilin-2 evaluated by SDS-PAGE and Western blot, respectively. The effect of alterations in these proteins on RBC morphology was evaluated by digital holographic microscopy and anionic transport efficiency of B3p was evaluated as sulphate uptake. Finally, PS- bearing MVs were quantified by annexin-V binding using FACS analysis. Findings presented in this paper indicate that RBC exposure to HgCl2 induces modifications in the electrophoretic profile of membrane protein fraction. Furthermore, our study reveals the Hg induced alterations of specific membrane proteins, such as Ankyrin, a protein essential for membrane-cytoskeleton linkage and Flotillin-2, a major integral protein of RBC lipid rafts, likely responsible for decreased membrane stability and increased fragmentations. Accordingly, under the same experimental conditions, RBC morphological changes and PS-bearing MVs release are observed. Finally, RBC treatment significantly affects the B3p-mediated anionic transport, that we report reduced upon HgCl2 treatment in a dose dependent manner. Altogether, the findings reported in this paper confirm that RBC are particularly vulnerable to Hg toxic effect and provide new insight in the Hg-induced protein modification in human RBC affecting the complex biological system of cellular membrane. In particular, Hg could induce dismantle of vertical cohesion between the plasma membrane and cytoskeleton as well as destabilization of lateral linkages of functional domains. Consequently, decreased membrane deformability could impair RBC capacity to deal with the shear forces in the circulation increasing membrane fragmentations. Furthermore, findings described in this paper have also significant implication in RBC physiology, particularly related to gas exchanges.

Andrographolide, isolated from Andrographis paniculata, induces apoptosis in monocytic leukemia and multiple myeloma cells via augmentation of reactive oxygen species production.

Background: Andrographolide (Andro) is a diterpenoid component of the plant Andrographis paniculata that is known for its anti-tumor activity against a variety of cancer cells. Methods: We studied the effects of Andro on the viability of the human leukemia monocytic cell line THP-1 and the human multiple myeloma cell line H929. Andro was compared with cytosine arabinoside (Ara-C) and vincristine (VCR), which are well-established therapeutics against hematopoietic tumors. The importance of reactive oxygen species (ROS) production for the toxicity of each agent was investigated by using an inhibitor of ROS production, N-acetyl-L-cysteine (NAC). Results: Andro reduced the viability of THP-1 and H929 in a concentration-dependent manner. H929 viability was highly susceptible to Andro, although only slightly susceptible to Ara-C. The agents Andro, Ara-C, and VCR each induced apoptosis, as shown by cellular shrinkage, DNA fragmentation, and increases in annexin V-binding, caspase-3/7 activity, ROS production, and mitochondrial membrane depolarization. Whereas Ara-C and VCR increased the percentages of cells in the G0/G1 and G2/M phases, respectively, Andro showed little or no detectable effect on cell cycle progression. The apoptotic activities of Andro were largely suppressed by NAC, an inhibitor of ROS production, whereas NAC hardly affected the apoptotic activities of Ara-C and VCR. Conclusions: Andro induces ROS-dependent apoptosis in monocytic leukemia THP-1 and multiple myeloma H929 cells, underlining its potential as a therapeutic agent for treating hematopoietic tumors.The high toxicity forH929 cells, by a mechanism that is different from that of Ara-C and VCR, is encouraging for further studies on the use of Andro against multiple myeloma.

Evaluation of eryptosis in patients with chronic kidney disease.

Anemia in patients with chronic kidney disease (CKD) is the result of reduced erythropoietin, disturbed erythropoiesis and decreased lifespan of circulating erythrocytes. Excessive eryptosis or premature suicidal erythrocyte death is characterized by cell shrinkage and phosphatidylserine externalization. This study aimed to explore accelerated eryptosis and accompanying biochemical alterations in CKD patients. A total of 106 CKD patients (59 predialysis [PreD] patients, 26 haemodialysis [HD] patients and 21 peritoneal dialysis [PD] patients) and a control group composed of 29 healthy volunteers were included in this study. Data on superoxide dismutase (SOD) activity (U/mL), annexin-V binding (mean fluorescent intensity, MFI) and intracellular calcium ([Ca2+]i; MFI) as well as the hematologic and biochemical parameters were recorded. The [Ca2+]i levels were 3.05 ± 1.66 MFI, 2.24 ± 0.99 MFI, 2.38 ± 0.87 MFI and 1.71 ± 0.46 MFI in the PreD, HD, PD and control groups, respectively. Other than significantly higher [Ca2+]i levels in the PreD group than in the control group (p < 0.001), no significant difference was noted between study groups in terms of [Ca2+]i. Annexin-V binding was 1.05 ± 0.99 MFI in PreD group, 1.15 ± 0.56 MFI in HD group, 1.06 ± 0.87 MFI in PD group, and 0.88 ± 0.86 MFI in controls. Annexin-V binding was significantly higher in PreD, HD and PD groups compared with the control group (p < 0.001 for each). SOD activity was 0.07 ± 0.07 in the PreD group, 0.13 ± 0.08 in the HD group, 0.14 ± 0.07 in the PD group, and 0.03 ± 0.01 in the control group. SOD activity in both HD and PD groups were significantly higher than control and PreD groups (p < 0.001 for each). Lower albumin, higher ferritin, and higher parathormon levels were found to be correlated with eryptosis biomarkers. Patients treated vs. non-treated with calcium channel blockers had significantly lower annexin-V binding levels (p = 0.013). Patients treated vs. non-treated with erythropoietin (EPO) had elevated annexin-V binding level (p < 0.001) and lower [Ca2+]i (p = 0.014). In conclusion, our findings revealed the presence accelerated eryptosis, as a potential contributing factor to development of anemia, in patients with CKD stages 3-5D. Inflamation and parathormon can also accelerate eryptosis. Favorable effect of CCB and EPO on eryptosis needs to be confirmed in larger scale studies.

Changes in glycans on platelet microparticles released during storage of apheresis platelets are associated with phosphatidylserine externalization and phagocytosis.

Platelets shed platelet microparticles (PMP) when activated or stored. As the removal of sialic acid (desialylation) promotes platelet uptake and clearance from the circulation, similar mechanisms for PMP uptake were hypothesized. The aim of the study was to investigate the role of surface glycans in the in vitro uptake of PMP from stored platelet components. Apheresis platelet components were stored in 40% plasma/60% SSP+ and sampled on day 1, 5, and 7 post-collection. PMP were characterized by staining with annexin-V (AnV) for phosphatidylserine (PS)-exposure, CD41 antibody, and fluorescently labeled glycan-binding lectins using flow cytometry. The procoagulant function of PMP following desialylation by neuraminidase treatment was assessed by AnV binding and a procoagulant phospholipid assay. PMP were isolated and stained with Deep Red, and phagocytosis by HepG2 cells was measured. Isolated PMP were deglycosylated with neuraminidase and galactosidase to assess the involvement of glycans in mediating phagocytosis. While the overall platelet surface glycan profile was unchanged during storage, PS+ platelets were sialylated, indicating different glycoproteins were changed. In contrast, sialic acid was removed from PS+ and CD41+ PMP, which specifically lost α-2,3-linked sialic acid during platelet storage. PMP were phagocytized by HepG2 cells, and PMP from platelets stored for 7 days were phagocytized to a lesser extent than on day 1. Desialylation by neuraminidase induced PS-exposure on PMP, decreased PPL clotting time, and increased PMP phagocytosis. PMP glycans change during platelet storage. Desialylation influences the procoagulant function of PMP and phagocytosis by HepG2 cells.

FLUORESCEIN LABELLED ANNEXIN V STAINING LIMITATIONS FOR DETERMINATION OF BLASTODERMAL CELLS APOPTOSIS

It is known that blastoderm contains not only blastodermal cells (BCs) but also cellular “debris”. These particles include yolk granules and lipid droplets. Some authors used AnnexinV (AnV) staining technique for determination of cell apoptosis. However, AnV is a protein with high affinity to lipids. So the aim of our study was to demonstrate the AnV binding in the blastoderm and yolk cell suspensions. Cell populations were obtained from fertilized chicken eggs. The blastoderms were isolated using filter paper ring method and mechanically fragmented. After that, samples were stained with AnV-FITC and analyzed by fluorescent microscopy (FM) and flow cytometry (FC). FM showed non-specific binding of AnV-FITC on the yolk and lipid particles. Therefore, the FC method using specific nuclear dye DRAQ5 was used to separate BCs from cellular debris. Only 1.82 ± 0.27 % cells were DRAQ5 positive. On the other hand, increase of total events (21 ± 3.30 %) of AnV positive cells without DRAQ5 dye was recorded. Moreover, the affinity of yolk to AnV by FC was also observed (10.45 ± 2.59 %). Significant differences among the groups (P &lt; 0.001) were recorded. This trend was caused by non-specific binding of AnV to the lipid and yolk particles. So it is necessary to stain suspension with nuclear dye to separate BCs than co-stained with some marker of apoptosis. Based on the results we can summarized that AnV staining is inappropriate technique for apoptosis of BCs, however, using nuclear dye we can prevent of non-specific binding.

The mechanism of the cytotoxic effect of Panax notoginseng extracts on prostate cancer cells

IntroductionPanax notoginseng (Burkill) F.H. commonly referred to as Sanqi, is a Chinese herb that has long been used to treat various conditions including blood disorders and cardiovascular diseases. While Panax notoginseng has been used as an anti-cancer medicinal herb in recent years, how it achieves this therapeutic effect has not been thoroughly elucidated. The purpose of this study was to reveal more about the mechanism of the cytotoxic effect of Panax notoginseng on prostate cancer (PCa) cells. MethodsEthanol extract of Panax notoginseng root was authenticated using high-performance liquid chromatography (HPLC). The cytotoxic activity of this herb against PCa cells was determined using the 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide (MTT) method, flow cytometry, and enzyme-linked immunosorbent assay (ELISA). ResultsThe assessment of cellular metabolic activity demonstrated that Panax notoginseng reduces the viability of LNCaP and 22Rv1 cells in a dose-dependent manner. Annexin-V binding flow cytometry assay showed that Panax notoginseng induces apoptosis in PCa cells. Cell cycle analysis by quantification of DNA content using flow cytometry showed that Panax notoginseng arrests the cell cycle at the G2/M phase in both LNCaP and 22Rv1 cells. Moreover, ELISA demonstrated that Panax notoginseng-treated PCa cells secrete significantly less tumor-promoting cytokine interleukin-4 (IL-4) to the supernatant compared with controls. ConclusionsThese results provide evidence for the cytotoxic effects of Panax notoginseng on PCa cell lines. This botanical is a promising candidate for the complementary and integrative medicine treatment of PCa and further studies are indicated to determine the anti-cancer mechanism of Panax notoginseng.

Design, synthesis, evaluation of new 3-acetylisoxazolines and their hybrid analogous as anticancer agents: In vitro and in silico analysis

3-Acetylisoxazolines were synthesized by the reaction of natural (R)-limonene and (R)-carvone with acetone in the presence of iron (III) nitrate. The reaction showed to be highly peri- and regioselective. Next, using a 1,3-dipolar cycloaddition reaction, the mono-3-acylisoxazolines derived from these monoterpenes were evaluated for their reactivity with nitrilimines. Only the enone of carvone-isoxazoline was regioselectively reactive, providing a new fused isoxazoline-carvone-pyrazolines. The structure of all the newly synthesized mono-cycloadducts (3 &5) and bis-cycloadducts (4 &7a–c) were fully identified based on their HRMS and NMR spectral data. They have also been screened for their cytotoxic activity against four human cancer cell lines: fibrosarcoma (HT-1080), lung carcinoma (A-549), and breast (MCF-7 and MDA-MB-231) cell lines. The obtained results showed that compound 4 was a potent cytotoxic agent against all selected cells. The possible mechanism of apoptosis induction by compound 4 was investigated using Annexin-V binding assay, caspase-3/7 activity and analysis cell cycle progression. The compound 4 induced the early apoptosis of both MCF-7 and MDA-MB-231 through caspase-3/7 activation, and the compound 4 have elicited S and G2/M phase arrest in MCF-7and MDA-MB-231 cancer cells, respectively. For further target investigations, a molecular docking study was employed and it showed that compound 4 has an inhibitory activity against Pim-1 protein kinase. Molecular dynamics study showed that compound 4/Pim-1 complex was stable during the simulation run at different time intervals. In-Silico ADMET predicted that compound 4 has good pharmacokinetic properties with high estimated oral bioavailability.