Annexin-positive Cells Research Articles

The role of ubiquitin and heat shock proteins 27 and 70 in the oxidative modification of proteins and the implementation of dexamethasone-induced apoptosis of tumor cells

BACKGROUND: Experimental studies of molecular control of the tumor cell’s redox status, which influences the implementation of apoptosis, are relevant for studying the pathogenesis of tumor growth.
 AIM: Studying the molecular mechanisms of the participation of ubiquitin and heat shock proteins 27 and 70 in the oxidative modification of proteins, amino acids and the implementation of dexamethasone-induced apoptosis of Jurkat tumor cells in conditions of decreased antioxidant protection by blocking the synthesis of reduced glutathione.
 MATERIAL AND METHODS: The effect of the inhibitor of de novo glutathione synthesis buthionine sulfoximine at a final concentration of 1 mM and/or the apoptosis inducer dexamethasone at a final concentration of 10 μM on the content of hydroxyl radical, protein-bound glutathione, carbonyl derivatives of proteins, oxidized tryptophan and bityrosine, ubiquitin, heat shock proteins 27 and 70, number of annexin V-positive cells and caspase-3 activity in Jurkat tumor cells was studied. Using the Shapiro–Wilk test, the normality of the distribution of indicators was assessed. Statistical hypotheses about the differences between the study groups were tested using the nonparametric Mann–Whitney test with a Bonferroni correction; correlation analysis was performed using the Spearman method at a significance level of p 0.05.
 RESULTS: In tumor cells of the Jurkat line, exposure to buthionine sulfoximine and dexamethasone was accompanied by a statistically significant decrease in the content of ubiquitin by 24% (p=0.004), protein-bound glutathione by 93% (p=0.003), oxidized tryptophan by 57% (p=0.003), and heat protein shock 70 by 56% (p=0.004), as well as an increase in the concentration of carbonyl derivatives of proteins by 53% (p=0.004), heat shock protein 27 by 104% (p=0.004), associated with an increase in the number of annexin-positive cells by 1296% (p=0.006) and caspase-3 activity by 258% relative to the values in intact cells. The relationship between an increase in the number of annexin-positive cells and caspase-3 activity with changes in the content of protein-bound glutathione, carbonylated proteins, oxidized tryptophan, ubiquitin and heat shock proteins 27 and 70 in tumor cells with simultaneous exposure to both buthionine sulfoximine and dexamethasone has been proven.
 CONCLUSION: Blocking de novo glutathione synthesis and stimulating apoptosis causes activation of reversible and irreversible oxidative modification of proteins and amino acids against the background of increased oxidative stress in Jurkat tumor cells.

Effect of pinocembrin on thymocyte proliferation and death

Introduction: Cell volume regulation is critical for cellular proliferation and death. Pinocembrin effectively suppresses the volume regulation in thymocytes under hypoosmotic stress by blocking the volume-sensitive anion channel. This study aims to evaluate the effects of this flavonoid on thymocyte proliferation and death. Methods: Thymocytes were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, and the cell number was determined by cloud-based automated cell counting (Corning). Necrotic and apoptotic cell death were evaluated by propidium iodide- and annexin V-staining, respectively. Results: Pinocembrin at 10–50 μM caused suppression of primary cultured thymocyte proliferation with a half-maximal effect of 28.4 ± 0.2 μM. The cell counts did not fall below the control level at the doses of 100–150 μM. The fraction of spontaneously necrotic cells was ~26% of the total population and increased to ~51% in the presence of dexamethasone. The fraction of spontaneously apoptotic cells increased by this glucocorticoid from 3.6% to 16.7%. Pinocembrin protected thymocytes from necrosis both in spontaneous and dexamethasone-induced death, reducing the fraction of necrotic cells by ~40–50% at 150 μM. Pinocembrin attenuated dexamethasone-induced apoptotic death, reducing the fraction of annexin-positive cells to the control (spontaneous) level. Conclusion: Our results suggest that pinocembrin arrests thymocyte proliferation without essential killing. Under conditions of massive death (e.g., during inflammation, when the level of glucocorticoids increases sharply both physiologically and as a result of pharmacotherapy), pinocembrin protects immuno-competent cells from necrotic and apoptotic death.

Myricetin-induced suicidal erythrocyte death.

Myricetin, a type of flavonol commonly found in fruits and herbs, has demonstrated anticancer properties by triggering the process of apoptosis or programmed cell death in tumor cells. Despite the absence of mitochondria and nuclei, erythrocytes can undergo programmed cell death, also known as eryptosis.This process is characterized by cell shrinkage, externalization of phosphatidylserine (PS) on the cell membrane, and the formation of membrane blebs. The signaling of eryptosis involves Ca2+ influx, the formation of reactive oxygen species (ROS), and the accumulation of cell surface ceramide. The present study explored the effects of myricetin on eryptosis. Human erythrocytes were exposed to various concentrations of myricetin (2-8 µM) for 24h. Flow cytometry was used to assess the markers of eryptosis, including PS exposure, cellular volume, cytosolic Ca2+ concentration, and ceramide accumulation. In addition, the levels of intracellular ROS were measured using the 2',7'-dichlorofluorescin diacetate (DCFDA) assay. The myricetin-treated (8 µM) erythrocytes significantly increased Annexin-positive cells, Fluo-3 fluorescence intensity, DCF fluorescence intensity, and the accumulation of ceramide. The impact of myricetin on the binding of annexin-V was significantly reduced, but not completely eliminated, by the nominal removal of extracellular Ca2+. Myricetin triggers eryptosis, which is accompanied and, at least in part, caused by Ca2+ influx, oxidative stress and increase of ceramide abundance.

The Nitrated Form of Nateglinide Induces Apoptosis in Human Pancreatic Cancer Cells Through a Caspase-dependent Mechanism.

Nitric oxide (NO) has antitumor activity against various solid tumor cell types in addition to its vasodilatory effect. In the current study, we investigated the effect and mechanism of the synthetic nitrated form (NO2-NAT) of nateglinide, a hypoglycemic agent, on the induction of cell death in human pancreatic cancer cells. NO production was evaluated by measuring nitrite (NO2) and nitrate (NO3) (NOx). Apoptotic cell numbers were determined using annexin V. NO2-NAT released nitrate and nitrite ions immediately upon dissolving in aqueous solution. NO2-NAT caused significant extracellular leakage of lactate dehydrogenase (LDH) in the human pancreatic cancer cell lines AsPC1 and BxPC3, and increased annexin-positive cells in a time- and concentration-dependent manner. NO2-NAT also significantly increased the activity of caspases 3 and 7. Exposure to Z-VAD-FMK, a caspase inhibitor, significantly suppressed NO2-NAT-induced cell death. These results indicated that NO2-NAT induces apoptosis in human pancreatic cancer cells and may serve as a new NO-based chemotherapeutic agent for the treatment of pancreatic cancer.

Thymol protects against 6-hydroxydopamine-induced neurotoxicity in in vivo and in vitro model of Parkinson\u2019s disease via inhibiting oxidative stress

BackgroundParkinson’s disease (PD) is a multifactorial movement disorder with the progressive degeneration of the nigrostriatal system that impairs patients’ movement ability. Oxidative stress has been found to affect the etiology and pathogenesis of PD. Thymol, a monoterpenic phenol, is one of the most important dietary constituents in thyme species. It has been used in traditional medicine and possesses some properties including antioxidant, free radical scavenging, anti-inflammatory. In this study, in vitro and in vivo experiments were performed with the thymol in order to investigate its potential neuroprotective effects in models of PD.MethodsThe present study aimed to evaluate the therapeutic potential of thymol in 6-hydroxydopamine (6-OHDA)-induced cellular and animal models of PD. ResultsPost-treatment with thymol in vitro was found to protect PC12 cells from toxicity induced by 6-OHDA administration in a dose-dependent manner by (1) increasing cell viability and (2) reduction in intracellular reactive oxygen species, intracellular lipid peroxidation, and annexin-positive cells. In vivo, post-treatment with thymol was protective against neurodegenerative phenotypes associated with systemic administration of 6-OHDA. Results indicated that thymol improved the locomotor activity, catalepsy, akinesia, bradykinesia, and motor coordination and reduced the apomorphine-caused rotation in 6-OHDA-stimulated rats. Increased level of reduced glutathione content and a decreased level of MDA (malondialdehyde) in striatum were observed in the 6-OHDA rats post-treated with thymol.ConclusionsCollectively, our findings suggest that thymol exerts protective effects, possibly related to an anti-oxidation mechanism, in these in vitro and in vivo models of Parkinson’s disease.

Anticancer Effects of Urtica Dioica in Breast Cancer.

Objective:The goal of this study is to look into the antiproliferative capabilities of Urtica Dioica (UD) on breast cancer. Methods:The cytotoxicity of UD extracts against breast cancer cell lines was investigated. Flow cytometry analyses were used to investigate in vitro apoptosis of breast cancer cells using Annexin V labeling. In vivo tests also performed.Results:UD showed cytotoxicity to three cancer cell lines. The number of Annexin-positive cells was higher in UD-treated cell lines than in untreated control cells. When compared to the untreated control group, the rats treated with UD had greater expressions of caspase 3, p53 protein, and TUNEL positive cells. When compared to the control group, Ki-67 expression was reduced in the treatment groups. In vivo tests revealed that, when compared to untreated rats, the mean tumor volume inhibition ratio in the UD group was 38 percent. Conclusion:These findings suggest that Urtica Dioica may have antitumoral properties in the treatment of breast cancer.

Calcium-oxidative stress signaling axis and casein kinase 1α mediate eryptosis and hemolysis elicited by novel p53 agonist inauhzin

Inauhzin (INZ) is a novel p53 agonist with antitumor activity. Anemia is a common side effect of chemotherapy and may arise from red blood cell (RBC) hemolysis or eryptosis. In this study, we investigate the mechanisms of INZ toxicity in human RBCs. RBCs were isolated from healthy donors and treated with antitumor concentrations of INZ (5–500 μM) for 24 h at 37 °C. Hemoglobin was photometrically measured, and cells were stained with Annexin-V-FITC for phosphatidylserine (PS), Fluo4/AM for calcium, and 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) for oxidative stress. INZ caused significant dose-responsive, calcium-dependent hemolysis starting at 40 μM. Furthermore, INZ significantly increased Annexin-positive cells and Fluo4 and DCF fluorescence. The cytotoxicity of INZ was also significantly mitigated in presence of D4476. INZ possesses hemolytic and eryptotic potential characterized by cell membrane scrambling, intracellular calcium overload, cell shrinkage, and oxidative stress secondary to calcium influx from the extracellular space.

Combination therapy using TGF-β1 and STI-571 can induce apoptosis in BCR-ABL oncogene-expressing cells.

The BCR-ABL oncogene is a tyrosine kinase gene that is over-expressed in CML. It inhibits the TGF-β1 signaling pathway. Due to resistance of cells to the tyrosine kinase inhibitor, STI-571, the combined effect of STI-571 and TGF-β1 on K562 cells was studied in the present research. Results revealed that the TGF-β1 cell signaling pathway, which is activated in K562 cells treated with TGF-β1, activates collective cell signaling pathways involved in survival and apoptosis. It is noteworthy that treating K562 cells with STI-571 triggered apoptotic pathways, accompanied by a reduction in proteins such as Bcl-xL, Bcl-2, p-AKT, p-Stat5, p-FOXO3, and Mcl-1 and an increase in the pro-apoptotic proteins PARP cleavage, and p27, leading to an increase in sub-G1 phase-arrested and Annexin-positive cells. Interestingly, the proliferation behavior of TGF-β1-induced cells was changed with the combination therapy, and STI-571-induced apoptosis was also prompted by this combination. Thus, combination treatment appears to promote sub-G1 cell cycle arrest compared to individually treated cells. Furthermore, it strongly triggered apoptotic signaling. In conclusion, TGF-β1 did not negatively impact the effect of STI-571, based on positive annexin cells, and AKT protein phosphorylation remains effective in apoptosis.

Assessment of Cytotoxic and Apoptotic Effects of Salvia syriaca L. in Colorectal Adenocarcinoma Cell Line (Caco-2).

This work is aimed to elucidate cytotoxic and apoptotic effects of Salvia syriaca essential oil and its chemical composition by GC-MS. The human colon cancer cells (Caco-2) were treated with different essential oil concentrations for 24 h. Crystal violet test was used to determine cell viability at 630 nm by using an ELISA reader. Apoptotic processes were measured by Annexin V-FITC Apoptosis Assay Kit. Germacrene D (21.77%), trans-β-ocimene (14.66%), β-pinene (9.07%), α-cadinol (8.19%) and α-pinene (6.50%) were the main components of oil determined by GC-MS. Moreover, we observed that the cytotoxic effect was increased with an increasing dose of essential oil. The EC50 value was calculated as 63.5 µg/mL. An increase in the percentage of apoptotic cells was observed after treatment of Caco-2 cells with S. syriaca essential oil revealed by image-based cytometry. A nearly 6-fold increase was found in annexin-positive cells after treatment. In terms of mRNA levels, RT-PCR analysis indicated that, although Bax and Caspase-3 were increased, Bcl-2 was decreased after oil treatment. According to our results, S. syriaca essential oil has promising phytochemicals that might be useful in cancer treatment due to their relatively cytotoxic and apoptotic activities in Caco-2 cells.

The Preclinical Activities of PTC596, a Novel Tubulin Binding Agent That Down-Regulates BMI1, Alone and in Combination with Bortezomib in Multiple Myeloma

Introduction: A novel tubulin binding agent PTC596, which is currently in clinical trials for solid tumors, was originally identified by its ability to kill cancer stem cells and to reduce BMI1 activity. PTC596 treatment results in hyperphosphorylation of the BMI1 protein and loss of BMI1 function as demonstrated by a reduction in H2A ubiquitination levels in a range of solid tumor lines. Subsequent studies have shown that the down-regulation of BMI1 protein is due to a G2/M arrest. In this study, we aimed to investigate the in-vitro and in-vivo anti-tumor activities of PTC596 and the combination with bortezomib in multiple myeloma (MM). Methods: For in-vitro evaluation, MTS and BrdU ELISA assays were performed using human MM cell lines. Approved by the Institutional Review Committee at Chiba University, primary myeloma cells and bone marrow stromal cells (BMSCs) were collected from the bone marrow of MM patients with informed consent. For in-vivo evaluation, the MM.1S subcutaneous xenograft model in NOG mice was used. To understand the mechanisms of action and target genes of the treatments, flow cytometry (FCM), western blotting, RNA-seq, and ChIP-seq were performed. Results: PTC596 induced significant cytotoxicity in all MM cell lines tested, including bortezomib-resistant OPM-2/BTZ and KMS-11/BTZ cells (CC50: 24-98 nM). PTC596 also suppressed cell proliferation when these cell lines were co-cultured with BMSCs. As expected, PTC596 reduced the levels of BMI1 protein and uH2A in a dose-dependent manner. Of note, PTC596 induced cell cycle arrest as detected by a BrdU FCM assay in MM cells and apoptosis as detected by annexin-V FCM in MM cell lines and primary myeloma cells. Moreover, oral administration of PTC596 twice a week for three weeks significantly inhibited the growth of MM.1S tumors implanted in immunodeficient mice and improved the survival of mice as compared with mice treated with vehicle only (p=0.0021). Of interest, bortezomib appeared to transcriptionally repress the expression of BMI1 and reduce the levels of uH2A. We then tested the efficacy of the combination of PTC596 with bortezomib in MM cells and found additive or synergistic effects when MM cell lines were co-cultured with BMSCs. Reductions in the levels of BMI1 protein and uH2A by PTC596 or bortezomib alone were significantly enhanced in the combination treatment. Furthermore, apoptosis induced by bortezomib was significantly enhanced by the combination with PTC596 as evidenced by increased annexin-positive cells detected in flow cytometric analysis and increased cleavage of caspases with reduction in MCL1 protein in western blotting. RNA-seq of MM.1S cells treated with PTC596 alone or in combination with bortezomib demonstrated repression of gene sets related to the cell cycle in either setting and enrichment of gene sets related to apoptosis in the combination. Ongoing analysis of our ChIP-seq data will reveal the direct targets of BMI1 in MM cells. Remarkably, oral administration of PTC596 combined with subcutaneous injection of bortezomib twice a week for five weeks significantly reduced MM.1S tumor growth in comparison to the control or either single treatment (p<0.0001 vs. control, p=0.0003 vs. PTC596, p=0.0104 vs. bortezomib). Severe body weight loss was not observed in the combination-treated mice except one mouse which needed washout period until recovery. White blood cell count and hemoglobin level in peripheral blood of mice were not significantly changed during the treatment with PTC596 or even the combination. Survival of the host mice treated with the combination was significantly prolonged as compared with the control or either single treatment (p<0.0001 vs. control, p=0.0002 vs. PTC596, p=0.0072 vs. bortezomib). Conclusions: Our results demonstrate that PTC596 alone and in combination with proteasome inhibition are potential novel therapeutic options in MM. The results of this study support the clinical evaluation of this promising therapeutic approach to improve the outcome of MM patients. Disclosures Sheedy: PTC Therapeutics: Employment. Weetall:PTC Therapeutics: Employment.

Carvacrol Protects Against 6-Hydroxydopamine-Induced Neurotoxicity in In Vivo and In Vitro Models of Parkinson’s Disease

Parkinson's disease (PD) is a progressive neurodegenerative movement disorder characterized by selective loss of dopaminergic neurons that project from the substantia nigra pars compacta to the striatum. Evidence from human and animal studies has suggested that oxidative damage critically contributes to neuronal loss in PD. Carvacrol (CAR), a monoterpenic phenol, is the main constituents in the essential oil of many aromatic plants and possesses some properties including anti-inflammatory and anti-oxidant effects. In this study, in vitro and in vivo experiments were performed with the CAR in order to investigate its potential neuroprotective effects in models of PD. Post-treatment with CAR in vitro was found to protect rat adrenal pheochromocytoma PC12 cells from toxicity induced by 6-hydroxydopamine (6-OHDA) administration in a dose-dependent manner by (1) increasing cell viability and (2) reduction in intracellular reactive oxygen species, intracellular lipid peroxidation, and annexin-positive cells. In vivo, post-treatment with CAR (15 and 20mg/kg) was protective against neurodegenerative phenotypes associated with systemic administration of 6-OHDA. Results indicated that CAR improved the locomotor activity, catalepsy, akinesia, bradykinesia, and motor coordination and reduced the apomorphine-caused rotation in 6-OHDA-stimulated rats. Increased level of reduced glutathione content and a decreased level of MDA (malondialdehyde) were observed in the 6-OHDA rats post-treated with CAR. These findings suggest that CAR exerts protective effects, possibly related to an anti-oxidation mechanism, in these in vitro and in vivo models of Parkinson's disease.

2121-P: nAChR Signaling Protects ß Cells from Apoptosis Induced by ER Stress and Specific Activation of IRE1α in INS1 Cells

Background: We previously showed that nAChR signaling regulates activation of IRE1α and markers of terminal-unfolded protein response (T-UPR) under endoplasmic reticulum stress (ERS) in β-cells. However, it is still unknown if nAChR signaling affects the apoptosis under ERS or specific activation of IRE1α in β-cells. We also previously reported that IRE1α specific inhibitor, KIRA-Kinase Inhibiting RNase Attenuator-, suppresses apoptosis induced by ERS in INS1 cells and β-cells in Akita mice, which suggest that IRE1α plays a critical role in ERS-induced apoptosis. The aim of this study is to determine if and how nAChR signaling regulates the apoptosis induced by ERS or specific activation of IRE1α in β-cells. Methods: We investigated the role of nAChR signaling on IRE1α signaling under ERS inducers or doxycycline-inducible IRE1α-overexpression (OE) in INS-1 cells. Cells were treated with Nicotine as an agonist of nAChR, and Tunicamycin or Thapsigargin as ERS inducers. Activation of IRE1α was confirmed by XBP1 mRNA splicing and phosphorylation of IRE1α. That of T-UPR was determined by Insulin1/proinsulin and TXNIP mRNA and protein expression. Translocation of phosphatidylserine to the external cell surface was detected by flow cytometry with Annexin V-FITC staining. Activation of caspase-3 and -7, markers of activation of caspase cascade, was detected by the active caspase-3/-7 specific substrate. Results: Nicotine reduced XBP1 mRNA splicing and phosphorylation of IRE1α induced by ERS. Nicotine reversed increase of TXNIP and decrease of Insulin1/proinsulin expressions induced by both ERS and IRE1α OE. Increased Annexin positive cells and caspase-3/7 activation by ERS were reduced by Nicotine. Finally, increased caspase-3/7 activation by IRE1α OE was reversed by Nicotine as well as KIRA. Conclusion: nAChR signaling could regulate IRE1α signaling and protect β-cells from apoptosis induced by ERS and specific activation of IRE1α in INS1 cells. Disclosure T. Ishibashi: None. S. Morita: None. A. Doi: None. H. Iwakura: None. H. Ariyasu: None. M. Nishi: None. H. Furuta: None. T. Akamizu: None.

Effect of Extracellular Vesicles Derived From Mesenchymal Stem Cells on K-562 Leukemia Cell Line

Background: Several studies show that mesenchymal stem cells (MSCs) possess anti-tumor properties, while other studies show that MSCs may promote cancer. Extracellular vesicles (EVs) are identified as novel mediators to communicate among cells, which may be used as vehicles to transfer the MSC information to the target cells. Objectives: The current study aimed at investigating whether the human umbilical cord mesenchymal stem cell (HUSCMSC) derived extracellular vesicles (EVs) inhibits or promotes tumor cell growth in K562 chronic myelogenous leukemia cell line. Methods: In the current experimental study, trypan blue exclusion and 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay were performed to investigate the cell viability and metabolic activity. Additionally, flow cytometry was employed to assess cell cycle and apoptosis of K562 cells after 72 hours exposure to EVs. Results: The obtained results showed that no doses of EVs inhibited the tumor growth (mean ± standard deviation (SD) of cell viability at the day 10 was 94.4 ± 2.60, P > 0.05) and metabolic activity in three days (mean ± SD at the day 3 was 97.27 ± 2.46, P > 0.05). Furthermore, EVs also did not change the apoptosis rate (mean ± SD at the day 3 was 3.17 ± 2.34 for annexin positive cells, and 3.36 ± 1.91 for annexin/propidium iodide (PI) -positive cells, P > 0.05). Conclusions: In conclusion, no significant changes in tumor cell growth were observed after treating K562 cells with MSCs-derived EVs.

Membrane permeability changes in radiation-induced apoptotic mouse thymocytes

Objective: Radiation-induced alterations in permeability of plasma membrane and associated apoptotic changes were investigated in apoptotic thymocytes induced by radiation. Materials and Methods: Immature mouse thymocytes suspended in RPMI 1640 were irradiated by γ-rays for desired doses up to 10 Gy and these cells were examined after incubation (up to 24 h) at 37°C. Radiation-induced changes in plasma membrane of thymocytes were determined by fluorescence technique using fluorescein diacetate (FDA) and trypan blue (TB) method. Induction of apoptosis in irradiated cells was determined using annexin-V as fluorescence probe method and measurement of cytosolic caspase-3 activity. Results: Radiation-induced apoptotic thymocytes showed an increase in membrane permeability as observed by leakage of FDA, which was poorly detected by TB. FDA could sensitively detect the dose-dependent variation in membrane permeability alterations in the range of 0.5–2 Gy incubated at 37°C. In radiation-induced apoptotic death, externalization of phosphatidylserine is an early event than caspase-3 activation. On incubation of cells, an increase in caspase-3 activity and proportion of annexin-positive cells was observed, which further increased after irradiation. Conclusion: Our results showed membrane permeability changes induced by γ-irradiation, which seem to be closely associated with the apoptosis in thymocytes.

2-Azetidinones: Synthesis and biological evaluation as potential anti-breast cancer agents

A series of twenty-five 2-azitidinone (β-lactam) derivatives were synthesized and evaluated for anti-cancer properties against breast cancer, MCF-7 and MDA-MB-231. These β-lactam derivatives depicted significant cytotoxicity in cancer cell lines but not in normal human mammary epithelial cells, MEpiC. Interestingly, derivatives of 2-bromo ethyl acrylonitrile (19w) exhibited - potent anti-proliferative activity with IC50, 5.79 ± 0.01 μM in MCF-7 and 6.86 ± 0.009 μM in MDA-MB-231. In addition, an increased expression of pro-apoptotic genes (p53, Bax, Bid) as well as decreased mRNA expression of cyclins D1, E and Cdk 2, 6 along with cell cycle arrest at G1 phase was observed. 19w treatment has shown higher percentage of Annexin-positive cells indicating induction of apoptosis. Further, docking studies confirmed an interaction between 19w and ATP-binding catalytic site of AKT1. Mechanistically, 19w depicted dose-dependent decrease in phosphorylation of AKT and GSK-3β and significant decrease in AKT kinase activity. In conclusion, β-lactam derivative 19w is a potential anti-breast cancer therapeutic candidate targeting cell survival pathway (AKT/GSK3β).

Preimplantation maternal stress impairs embryo development by inducing oviductal apoptosis with activation of the Fas system.

What are the mechanisms by which the preimplantation restraint stress (PIRS) impairs embryo development and pregnancy outcome? PIRS impairs embryo development by triggering apoptosis in mouse oviducts and embryos,and this involves activation of the Fas system. Although it is known that the early stages of pregnancy are more vulnerable than later stages to prenatalstress, studies on the effect of preimplantation stress on embryo developmentare limited. Furthermore, the mechanisms by which psychological stress impairs embryo development are largely unknown. These issues are worth exploring using the mouse PIRS models because restraint of mice is an efficient experimental procedure developed for studies of psychogenic stress. Mice of Kunming strain, the generalized lymphoproliferative disorder (gld) mice with a germline mutation F273L in FasL in a C57BL/6J genomic background and the wild-type C57BL/6J mice were used. Female and male mice were used 8-10 weeks and 10-12 weeks after birth, respectively. Female mice showing vaginal plugs were paired by weight and randomly assigned to restraint treatments or as controls. For restraint treatment, an individual mouse was put in a micro-cage with food and water available. Control mice remained in their cages with food and water during the time treated females were stressed. Female mice were exposed to PIRS for 48 h starting from 16:00 on the day of vaginal plug detection. At the end of PIRS, levels of glucorticoids (GC), corticotropin-releasing hormone (CRH)and redox potential were measured in serum, while levels of GC, GC receptor (GR), CRH, CRH receptor (CRHR), Fas and Fas ligand (FasL) protein, mRNAs for brain derived neurotrophic factor (BDNF) and insulin-like growth factor-1 (IGF-1), oxidative stress (OS) and apoptosis were examined in oviducts. Preimplantation development and levels of GR, Fas, redox potential and apoptosis were observed in embryos recovered at different times after the initiation of PIRS. The gld mice were used to confirm a role for the Fas system in triggering apoptosis of embryos and oviducts. Compared to those in control mice, while the number of blastocysts/mouse (5.0±0.7 versus 11.1±0.5), cell number/blastocyst (49.1±1.3 versus 61.5±0.9), percentages of term pregnancy (37.5% versus 90.9%) and litter size (3.7±0.1versus 9.6±0.6) decreased, blood CRH (560±23 versus 455±37 pg/ml), cortisol (27.3±3.4 versus 5±0.5 ng/ml) and OS index (OSI: 2.8 versus 1.7) increased significantly (all P<0.05) following PIRS. In the oviduct, while levels of CRH (1175±85 versus 881±33 pg/100 mg), cortisol (28.9±1.7 versus14±4 ng/g), CRHR (2.3±0.3 versus 1.0±0.0), FasL (1.31±0.06 versus 1.08±0.05 ng/g), Fas (1.42±0.13 versus 1.0±0.0) and apoptotic cells (19.1±0.5% versus 8.4±0.4%) increased, levels of GR proteins (0.67±0.14 versus 1.0±0.0) and Igf-1 (0.6±0.09 versus 1.0±0.0) and Bdnf (0.73±0.03 versus 1.0±0.0) mRNAs decreased significantly (all P<0.05 versus control) after PIRS. Mouse embryos expressed GR and Fas at all stages of preimplantation development and embryo OS (GSH/GSSG ratio: 0.88±0.03 versus 1.19±0.13) and annexin-positive cells (blastocysts: 31.4±3.8% versus 10.96±3.4%) increased significantly (P<0.05) following PIRS. Furthermore, the detrimental effects of PIRS on embryo development and oviductal apoptosis were much reduced in gld mice. Thus, PIRS triggered apoptosis in oviductal cells with activation of the Fas/FasL system. The apoptotic oviductal cells promoted embryo apoptosis with reduced production of IGF-1 and BDNF and increased production of FasL. Although important, the conclusions were drawn from limited results obtained using a single model in one species and thus they need further verification using other models and/or in other species. Furthermore, as differences in stressed samples were modest and sometimes not significant between gld and wild-type mice whereas differences between control and stressed samples were always present within gld mice, it is deduced that signaling pathways other than the Fas/FasL system might be involved as well in the PIRS-triggered apoptosis of oviducts and embryos. The data are important for studies on the mechanisms by which psychological stress affects female reproduction, as FasL expression has been observed in human oviduct epithelium. Not applicable. This study was supported by grants from the National Basic Research Program of China (Nos. 2014CB138503 and 2012CB944403), the China National Natural Science Foundation (Nos. 31272444 and 30972096) and the Animal breeding improvement program of Shandong Province. All authors declare that their participation in the study did not involve factual or potential conflicts of interests.

Growth inhibitory effects of Withaferin A on MDS-L cells – A human 5q Myelodysplastic Syndrome (MDS) cell line

Abstract Myelodysplastic syndrome (MDS) affects bone marrow production of white blood cells and erythrocytes. At least 40% of MDS patients who go into remission after treatment eventually progress to acute myelogenous leukemia (AML) which is very difficult to treat. Hence, there is a compelling need for new and better therapies. Withaferin A (WFA), a withanolide isolated from Winter cherry (Withania somnifera), has been shown to have potent anti-cancer properties in several animal models of cancer. We found that WFA inhibited the growth of MDS-L cells, a human cell line derived from a 5q MDS patient. Treatment with WFA induced apoptosis of MDS-L cells, indicated by the cleavage of caspase-3 as early as 6hrs after treatment and a dose dependent increase of annexin positive cells. Cell cycle analysis revealed that WFA caused MDS-L cells to arrest at S and G2/M and accordingly there was a reduction in cyclin A/CDK2 and cyclin B/CDK1 respectively. WFA also inhibited the activity of the heat shock protein (Hsp 90) chaperone as shown by the degradation of its client proteins such as Akt, an important pro-survival kinase and increased expression of Hsp70 which partially compensates for decrease in Hsp90 function. Interestingly, the mechanism by which WFA acts on MDS-L cells is independent of its well-recognized action on Nf-κB. Microarray analysis of gene expression in MDS-L cells showed a dramatic increase in several genes that may have a role in Nf-κB independent growth inhibitory effects of WFA. Studies with a xenograft model of MDS-L cells in NSGS mice suggest WFA might also be effective in vivo. The potent anti-proliferative and anti-survival effects of WFA on MDS-L cells, suggest that it could be a novel therapeutic agent for the treatment of 5q MDS.

MECHANISMS OF APOPTOSIS DYSREGULATION IN CANCER CELLS UNDER THE CONDITIONS OF REDOX STATUS MODULATION

Introduction. Changes in the redox status of tumor cells can be used as one of the molecular mechanisms of apoptosis aimed at increasing the susceptibility of tumor cells to chemotherapeutic agents. Purpose: to study the mechanisms of dysregulation of apoptosis in P19 tumor cells under the conditions of redox status modulation. Material and methods. Apoptosis in P19 tumor cells was assessed by flow cytometry analysis. The number of annexin-positive cells, the expression of CD95 and CD120, as well as the intracellular calcium ion concentration and the percentage of cells with reduced mitochondrial transmembrane potential were measured. The protein-glutathione mixed-disulfide level and the GSH/GSSG ratio were determined by spectrophotometry. To modulate redox status of cells, the protector and blocker of SH-groups, or N-acetylcysteine were used. Results. Incubation of cultures in the presence of SH-group blocker resulted in the imbalance in the glutathione system with increased concentration of glutathionylated proteins. A decreased redox status led to an increased CD95 and CD120 expression levels on the membrane of P19 tumor cells, as well as to decreased mitochondrial potential and increased intracellular calcium ion concentration, thus contributing to the launch of a P19 tumor cells. The presence of SH-group blocker and N-acetylcysteine resulted in an increased number of annexinpositive cells. Conclusion. Along with the development of oxidative stress, the molecular redox-dependent mechanisms of apoptosis dysregulation through the mitochondrial and receptor-mediated pathways were identified in the P19 tumor cells.

Pharmacogenomics of Bortezomib in Multiple Myeloma Patients Reveals That the Ubiquitin Ligase SCF-Skp2 Promotes Drug Resistance

The ability to couple cancer patients with genetically-targeted therapeutic strategies remains a major challenge due to the heterogeneous and evolvable nature of individual tumors. This is particularly true of the molecularly diverse and complex disease multiple myeloma (MM). Clinical efficacy of the proteasome inhibitor (PI) Bortezomib (VelcadeTM) established the Ub+proteasome system (UPS) as a key target in MM. While the therapeutic benefit of PIs in MM remains unchallenged, many patients do not respond to therapy and drug resistance develops nearly uniformly. Despite marked therapeutic advances for most patients with MM, outcomes remain poor for 15% to 20% of patients presenting with high-risk disease defined by host-related and tumor-related factors and expert guidelines recommend enrollment in a clinical trial for the majority of patients who will ultimately fail approved therapies.The Skp1-Cul1-F-box protein (SCF) ubiquitin ligase SCFSkp2 promotes ubiquitination and degradation of the cell-cycle inhibitor p27. Increased SKP2 expression and reduced p27 levels are frequent in human cancers and are associated with therapeutic resistance and poor prognosis. SCFSkp2 ubiquitination activity is increased by Commd1, a copper metabolism MURR1 domain-containing 1 protein that binds Cul1 and by Cks1B that increases binding of phosphorylated p27 to Skp2. Bortezomib is a proteasome inhibitor that has remarkably improved multiple myeloma (MM) patient outcome, but dose-limiting toxicities and development of resistance limit its long-term utility. Here, we observed higher CUL1 and COMMD1 mRNA levels in Bortezomib-resistant patient MM cells and correlated higher CUL1, COMMD1, SKP2 and CKS1B levels with decreased survival. Genetic knockdown of CUL1 or COMMD1 disrupted the SCFSkp2 complex, stabilized p27, synergistically increased the generation of annexin-positive cells by bortezomib and enhanced the anti-myeloma effect of PIs against resistant cells. Chemical library screening identified a novel compound, DT204, that reduced Skp2 association with Cul1 and Commd1, inhibited p27 degradation and induced synergistic anti-MM activity combined with Bortezomib in patient tumor cells and chemoresistant stem cell-like cells to overcame Bortezomib resistance. Bortezomib treatment with DT204 also reduced the growth of xenotransplanted myeloma cells in mouse models and prolongs host survival. Taken together, our results demonstrate that the SCFSkp2 -p27 axis represents a major determinant in the ability of Bortezomib to induce apoptosis in MM and provide proof-of-concept for the combination of SCFSkp2 inhibitors with Bortezomib as a strategy to overcome Bortezomib resistance. DisclosuresNo relevant conflicts of interest to declare.

Molecular mechanism of anti-cancer activity of phycocyanin in triple-negative breast cancer cells.

BackgroundTriple-negative breast cancers represent an important clinical challenge, as these cancers do not respond to conventional endocrine therapies or other available targeted agents. Phycocyanin (PC), a natural, water soluble and non-toxic molecule is shown to have potent anti-cancer property.MethodsIn this study, we determined the efficacy of PC as an anti-neoplastic agent in vitro on a series of breast cancer cell lines. We studied effects of PC in inducing DNA damage and apoptosis through western blot and qPCR. Also, anti-metastatic and anti-angiogenic properties were studied by classic wound healing and vasculogenic mimicry assays.ResultsWe found that triple negative MDA-MB-231 cells were most sensitive to PC (IC50 : 5.98 ± 0.95 μM) as compared to other cells. They also showed decreased cell proliferation and reduced colony formation ability upon treatment with PC. Profile of Cell cycle analysis showed that PC caused G1 arrest which could be attributed to decreased mRNA levels of Cyclin E and CDK-2 and increased p21 levels. Mechanistic studies revealed that PC induced apoptosis as evident by increase in percentage of annexin positive cells, increase in γ-H2AX levels, and by changing the Bcl-2/Bax ratio followed by release of cytochrome C and increased Caspase 9 levels. MDA MB 231 cells treated with PC resulted in decreased cell migration and increased cell adhesive property and also showed anti-angiogenic effects. We also observed that PC suppressed cyclooxygenase-2 (COX-2) expression and prostaglandin E(2) production. All these biological effects of phycocyanin on MDA MB 231 cells could be attributed to decreased MAPK signaling pathway. We also observed that PC is non-toxic to non-malignant cells, platelets and RBC’s.ConclusionTaken together, these findings demonstrate, for the first time, that PC may be a promising anti-neoplastic agent for treatment of triple negative breast cancers.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1784-x) contains supplementary material, which is available to authorized users.