Hyperthermia induces differentiation without apoptosis in permissive temperatures in human erythroleukaemia cells

Purpose: The aim of the present study was to investigate whether induction of differentiation by hyperthermia is accompanied by apoptosis and necrosis to further evaluate the benefits of using hyperthermia as a differentiation inducing physical modality. Materials and method: Differentiation was evaluated in K562 erythroleukaemia cells by measuring haemoglobin synthesis and flow cytometric measurement of glycophorin A expression. Apoptosis was measured by Annexin-V-FITC and Propidium Iodide (PI) double staining assay. Apoptosis and necrosis was also evaluated morphologically using staining with acridine orange/ethidium bromide (AO/EtBr) by fluorescence microscopy. Heat shock protein 70 (HSP70) level was measured by ELISA kit. Results: Hyperthermia (43°C) induced differentiation as judged by increased haemoglobin synthesis and glycophorin A expression. No sign of apoptosis or necrosis could be detected at this temperature. Cell viability did not change due to heat treatment, and cellular proliferation was reduced in a dose (heating time) dependent manner. At 45°C, hyperthermia induced apoptosis and necrosis with minimal or no sign of differentiation. HSP70 level was significantly increased at 43°C along with differentiation of leukaemic cells, while at 45°C no significant effect on HSP70 production could be observed. Conclusions: The encouraging results obtained here indicate that by heat treatment at 43°C, hyperthermia can be used alone or in combination with other modalities as a differentiation inducing agent without any detectable apoptotic activity. Positive correlation between HSP70 production and induction of differentiation and lack of apoptosis by hyperthermia confirm the possible role of HSP70 in the heat-induced differentiation and apoptosis in leukaemic cells

Thermal Unfolding Pathway of PHD2 Catalytic Domain in Three Different PHD2 Species: Computational Approaches

Prolyl hydroxylase domain 2 containing protein (PHD2) is a key protein in regulation of angiogenesis and metastasis. In normoxic condition, PHD2 triggers the degradation of hypoxia-inducible factor 1 (HIF-1α) that induces the expression of hypoxia response genes. Therefore the correct function of PHD2 would inhibit angiogenesis and consequent metastasis of tumor cells in normoxic condition. PHD2 mutations were reported in some common cancers. However, high levels of HIF-1α protein were observed even in normoxic metastatic tumors with normal expression of wild type PHD2. PHD2 malfunctions due to protein misfolding may be the underlying reason of metastasis and invasion in such cases. In this study, we scrutinize the unfolding pathways of the PHD2 catalytic domain’s possible species and demonstrate the properties of their unfolding states by computational approaches. Our study introduces the possibility of aggregation disaster for the prominent species of PHD2 during its partial unfolding. This may justify PHD2 inability to regulate HIF-1α level in some normoxic tumor types

A biophysical study on the mechanism of interactions of DOX or PTX with α-lactalbumin as a delivery carrier

© 2018, The Author(s). Doxorubicin and paclitaxel, two hydrophobic chemotherapeutic agents, are used in cancer therapies. Presence of hydrophobic patches and a flexible fold could probably make α-Lactalbumin a suitable carrier for hydrophobic drugs. In the present study, a variety of thermodynamic, spectroscopic, computational, and cellular techniques were applied to assess α-lactalbumin potential as a carrier for doxorubicin and paclitaxel. According to isothermal titration calorimetry data, the interaction between α-lactalbumin and doxorubicin or paclitaxel is spontaneous and the K (M−1) value for the interaction of α-lactalbumin and paclitaxel is higher than that for doxorubicin. Differential scanning calorimetry and anisotropy results indicated formation of α-lactalbumin complexes with doxorubicin or paclitaxel. Furthermore, molecular docking and dynamic studies revealed that TRPs are not involved in α-Lac’s interaction with Doxorubicin while TRP 60 interacts with paclitaxel. Based on Pace analysis to determine protein thermal stability, doxorubicin and paclitaxel induced higher and lower thermal stability in α-lactalbumin, respectively. Besides, fluorescence lifetime measurements reflected that the interaction between α-lactalbumin with doxorubicin or paclitaxel was of static nature. Therefore, the authors hypothesized that α-lactalbumin could serve as a carrier for doxorubicin and paclitaxel by reducing cytotoxicity and apoptosis which was demonstrated during our in vitro cell studies

Caffeic acid phenethyl ester increases radiosensitivity of estrogen receptor- positive and -negative breast cancer cells by prolonging radiation-induced DNA Damage

Purpose: Breast cancer is an important cause of death among women. The development of radioresistance in breast cancer leads to recurrence after radiotherapy. Caffeic acid phenethyl ester (CAPE), a polyphenolic compound of honeybee propolis, is known to have anticancer properties. In this study, we examined whether CAPE enhanced the radiation sensitivity of MDAMB- 231 (estrogen receptor-negative) and T47D (estrogen receptor- positive) cell lines. Methods: The cytotoxic effect of CAPE on MDA-MB-231 and T47D breast cancer cells was evaluated by performing an 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay. To assess clonogenic ability, MDAMB- 231 and T47D cells were treated with CAPE (1 μM) for 72 hours before irradiation, and then, a colony assay was performed. A comet assay was used to determine the number of DNA strand breaks at four different times. Results: CAPE decreased the viability of both cell lines in a dose- and time-dependent manner. In the clonogenic assay, pretreatment of cells with CAPE before irradiation significantly reduced the surviving fraction of MDA-MB-231 cells at doses of 6 and 8 Gy. A reduction in the surviving fraction of T47D cells was observed relative to MDA-MB-231 at lower doses of radiation. Additionally, CAPE maintained radiation-induced DNA damage in T47D cells for a longer period than in MDA-MB-231 cells. Conclusion: Our results indicate that CAPE impairs DNA damage repair immediately after irradiation. The induction of radiosensitivity by CAPE in radioresistant breast cancer cells may be caused by prolonged DNA damage. © 2016 Korean Breast Cancer Society. All rights reserved

Properties of poloxamer molecules and poloxamer micelles dissolved in water and next to lipid bilayers : results from computer simulations

To study the properties of poloxamer molecules P85 and P188 and micelles containing these poloxamers in bulk water and also next to lipid bilayers, we performed coarse-grained molecular dynamics computer simulations. We used MARTINI force-field and adjusted Lennard-Jones nonbonded interaction strength parameters for poloxamer beads to take into account the presence of polarizable water. Simulations of systems containing poloxamer molecules or micelles solvated in bulk water showed that structural properties, such as radii of gyration of the molecules and micelles, agree with the ones inferred from experiments. We observed that P85 micelle is almost spherical in shape, whereas the P188 micelle is distorted from being spherical. Simulations containing systems with the water?lipid bilayer interface showed that hydrophilic blocks of poloxamers interact with lipid headgroups of the bilayer and remain at the interface, whereas hydrophobic blocks prefer to insert into the central hydrophobic region of the bilayer. Simulations containing poloxamer micelles next to lipid bilayer showed no permeation of these micelles into the bilayer. To study the ?healing? properties of P188 poloxamer, we performed simulations on a system containing a P188 micelle next to ?damaged? lipid bilayer containing a pore. We observed that hydrophobic chains of poloxamers got inserted into the bilayer through the pore region, ultimately closing the pore

Distinct Dynamics of Migratory Response to PD-1 and CTLA-4 Blockade Reveals New Mechanistic Insights for Potential T-Cell Reinvigoration following Immune Checkpoint Blockade

Cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and programmed cell death protein 1 (PD-1), two clinically relevant targets for the immunotherapy of cancer, are negative regulators of T-cell activation and migration. Optimizing the therapeutic response to CTLA-4 and PD-1 blockade calls for a more comprehensive insight into the coordinated function of these immune regulators. Mathematical modeling can be used to elucidate nonlinear tumor–immune interactions and highlight the underlying mechanisms to tackle the problem. Here, we investigated and statistically characterized the dynamics of T-cell migration as a measure of the functional response to these pathways. We used a previously developed three-dimensional organotypic culture of patient-derived tumor spheroids treated with anti-CTLA-4 and anti-PD-1 antibodies for this purpose. Experiment-based dynamical modeling revealed the delayed kinetics of PD-1 activation, which originates from the distinct characteristics of PD-1 and CTLA-4 regulation, and followed through with the modification of their contributions to immune modulation. The simulation results show good agreement with the tumor cell reduction and active immune cell count in each experiment. Our findings demonstrate that while PD-1 activation provokes a more exhaustive intracellular cascade within a mature tumor environment, the time-delayed kinetics of PD-1 activation outweighs its preeminence at the individual cell level and consequently confers a functional dominance to the CTLA-4 checkpoint. The proposed model explains the distinct immunostimulatory pattern of PD-1 and CTLA-4 blockade based on mechanisms involved in the regulation of their expression and may be useful for planning effective treatment schemes targeting PD-1 and CTLA-4 functions

Enterolactone: A novel radiosensitizer for human breast cancer cell lines through impaired DNA repair and increased apoptosis

Introduction Radiotherapy is a potent treatment against breast cancer, which is the most commonly diagnosed cancer among women. However, the emergence of radioresistance due to increased DNA repair leads to radiotherapeutic failure. Applying polyphenols combined with radiation is a more promising method leading to better survival. Enterolactone, a phytoestrogenic polyphenol, has been reported to inhibit an important radioresistance signaling pathway, therefore we conjectured that enterolactone could enhance radiosensitivity in breast cancer. To assess this hypothesis, radiation response of enterolactone treated MDA-MB-231 and T47D cell lines and corresponding cellular mechanisms were investigated. Methods Cytotoxicity of enterolactone was measured via MTT assay. Cells were treated with enterolactone before X-irradiation, and clonogenic assay was used to evaluate radiosensitivity. Cell cycle distribution and apoptosis were measured by flow cytometric analysis. In addition, DNA damages and corresponding repair, chromosomal damages, and aberrations were assessed by comet, micronucleus, and cytogenetic assays, respectively. Results Enterolactone decreased the viability of cells in a concentration- and time dependent manner. Enterolactone significantly enhanced radiosensitivity of cells by abrogating G2/M arrest, impairing DNA repair, and increasing radiation-induced apoptosis. Furthermore, increased chromosomal damages and aberrations were detected in cells treated with enterolactone combined with X-rays than X-ray alone. These effects were more prominent in T47D than MDA-MB-231 cells. Discussion To our knowledge, this is the first report that enterolactone is a novel radiosensitizer for breast cancer irrespective of estrogen receptor status. Authors propose enterolactone as a candidate for combined therapy to decrease the radiation dose delivered to patients and subsequent side effects. © 2016 Elsevier Inc

Thermal Unfolding Pathway of PHD2 Catalytic Domain in Three Different PHD2 Species: Computational Approaches

Prolyl hydroxylase domain 2 containing protein (PHD2) is a key protein in regulation of angiogenesis and metastasis. In normoxic condition, PHD2 triggers the degradation of hypoxia-inducible factor 1 (HIF-1a) that induces the expression of hypoxia response genes. Therefore the correct function of PHD2 would inhibit angiogenesis and consequent metastasis of tumor cells in normoxic condition. PHD2 mutations were reported in some common cancers. However, high levels of HIF-1a protein were observed even in normoxic metastatic tumors with normal expression of wild type PHD2. PHD2 malfunctions due to protein misfolding may be the underlying reason of metastasis and invasion in such cases. In this study, we scrutinize the unfolding pathways of the PHD2 catalytic domain’s possible species and demonstrate the properties of their unfolding states by computational approaches. Our study introduces the possibility of aggregation disaster for the prominent specie