Mesenchymal stem cells induce PD-L1 expression through the secretion of CCL5 in breast cancer cells.

Various factors in the tumor microenvironment (TME) regulate the expression of PD-L1 in cancer cells. In TME, mesenchymal stem cells (MSCs) play a crucial role in tumor progression, metastasis, and drug resistance. Emerging evidence suggests that MSCs can modulate the immune-suppression capacity of TME through the stimulation of PD-L1 expression in various cancers; nonetheless, their role in the induction of PD-L1 in breast cancer remained elusive. Here, we assessed the potential of MSCs in the stimulation of PD-L1 expression in a low PD-L1 breast cancer cell line and explored its associated cytokine. We assessed the expression of MSCs-related genes and their correlation with PD-L1 across 1826 breast cancer patients from the METABRIC cohort. After culturing an ER+/differentiated/low PD-L1 breast cancer cells with MSCs conditioned-medium (MSC-CM) in a microfluidic device, a variety of in-vitro assays was carried out to determine the role of MSC-CM in breast cancer cells' phenotype plasticity, invasion, and its effects on induction of PD-L1 expression. In-silico analysis showed a positive association between MSCs-related genes and PD-L1 expression in various types of breast cancer. Through functional assays, we revealed that MSC-CM not only prompts a phenotype switch but also stimulates PD-L1 expression at the protein level through secretion of various cytokines, especially CCL5. Treatment of MSCs with cytokine inhibitor pirfenidone showed a significant reduction in the secretion of CCL5 and consequently, expression of PD-L1 in breast cancer cells. We concluded that MSCs-derived CCL5 may act as a PD-L1 stimulator in breast cancer.

Direct and indirect effects of exposure to 900 MHz GSM radiofrequency electromagnetic fields on CHO cell line: Evidence of bystander effect by non-ionizing radiation.

INTRODUCTION: The rapid rise in global concerns about the adverse health effects of exposure to radiofrequency radiation (RFR) generated by common devices such as mobile phones has prompted scientists to further investigate the biological effects of these environmental exposures. Non-targeted effects (NTEs) are responses which do not need a direct exposure to be expressed and are particularly significant at low energy radiations. Although NTEs of ionizing radiation are well documented, there are scarcely any studies on non-targeted responses such as bystander effect (BE) after exposure to non-ionizing radiation. The main goal of this research is to study possible RFR-induced BE. MATERIAL AND METHODS: Chinese hamster ovary cells were exposed to 900 MHz GSM RFR at an average specific absorption rate (SAR) of 2 W/kg for 4, 12 and 24 hours (h). To generate a uniformly distributed electromagnetic field and avoid extraneous RF exposures a cavity was desined and used. Cell membrane permeability, cell redox activity, metabolic and mitotic cell death and DNA damages were analyzed. Then the most effective exposure durations and statistically significant altered parameters were chosen to assess the induction of BE through medium transfer procedure. Furthermore, intra and extra cellular reactive oxygen species (ROS) levels were measured to assess the molecular mechanism of BE induced by non-ionizing radiation. RESULTS: No statistically significant alteration was found in cell membrane permeability, cell redox activity, metabolic cell activity and micronuclei (MN) frequency in the cells directly exposed to RFR for 4, 12, or 24 h. However, RFR exposure for 24 h caused a statistically significant decrease in clonogenic ability as well as a statistically significant increase in olive moment in both directly exposed and bystander cells which received media from RFR-exposed cells (conditioned culture medium; CCM). Exposure to RFR also statistically significant elevated both intra and extra cellular levels of ROS. CONCLUSION: Our observation clearly indicated the induction of BE in cells treated with CCM. To our knowledge, this is the first report that a non-ionizing radiation (900 MHz GSM RFR) can induce bystander effect. As reported for ionizing radiation, our results proposed that ROS can be a potential molecule in indirect effect of RFR. On the other hand, we found the importance of ROS in direct effect of RFR but in different ways.

Magnetic bio-metal-organic framework nanocomposites decorated with folic acid conjugated chitosan as a promising biocompatible targeted theranostic system for cancer treatment.

In this work, a multifunctional magnetic Bio-Metal-Organic Framework (Fe3O4@Bio-MOF) coated with folic acid-chitosan conjugate (FC) was successfully prepared for tumor-targeted delivery of curcumin (CUR) and 5-fluorouracil (5-FU) simultaneously. Bio-MOF nanocomposite based on CUR as organic linker and zinc as metal ion was prepared by hydrothermal method in the presence of amine-functionalized Fe3O4 magnetic nanoparticles (Fe3O4@NH2 MNPs). 5-FU was loaded in the magnetic Bio-MOF and the obtained nanocarrier was then coated with FC network. The prepared nanocomposite (NC) was fully characterized by high resolution-transmission electron microscope (HR-TEM), field emission scanning electron microscopy (FE-SEM), Dynamic light scattering (DLS), X-ray diffraction analysis (XRD), thermogravimetric analysis (TGA), vibrating sample magnetometry (VSM), nuclear magnetic resonance (NMR), and UV-vis analyses. In vitro release study showed controlled release of CUR and 5-FU in acidic pH confirming high selectivity and performance of the carrier in cancerous microenvironments. The selective uptake of 5-FU-loaded Fe3O4@Bio-MOF-FC by folate receptor-positive MDA-MB-231 cells was investigated and verified. The ultimate nanocarrier exhibited no significant toxicity, while drug loaded nanocarrier showed selective and higher toxicity against the cancerous cells than normal cells. SDS PAGE was also utilized to determine the protein pattern attached on the surface of the nanocarriers. In vitro and in vivo MRI studies showed negative signal enhancement in tumor confirming the ability of the nanocarrier to be applied as diagnostic agent. Owing to the selective anticancer release and cellular uptake, acceptable blood compatibility as well as suitable T2 MRI contrast performance, the target nanocarrier could be considered as favorable theranostic in breast cancer.

Theranostic α-Lactalbumin-Polymer-Based Nanocomposite as a Drug Delivery Carrier for Cancer Therapy.

A nanotheranostic system was developed using α-lactalbumin along with Fe3O4 nanoparticles as an magnetic resonance imaging (MRI) contrast agent for medical imaging and doxorubicin as the therapeutic agent. α-lactalbumin was precipitated and cross-linked using poly(ethylene glycol) and glutaraldehyde. Besides, polyethylenimine was applied to increase the number of amine groups during cross-linking between α-lactalbumin and Fe3O4 nanoparticles. Interestingly, 90% of the initial protein used for the coaggregation process was incorporated in the prepared 130 nm nanocomposites, which facilitated the 85% doxorubicin loading. Formation of pH-sensitive imine bonds between glutaraldehyde and amine groups on α-lactalbumin and polyethylenimine resulted in higher release of doxorubicin at acidic pHs and consequently development of a pH-sensitive nanocarrier. The designed nanocomposite was less immunogenic owing to stimulating the production of less amounts of C3a, C5a, platelet factor 4, glycoprotein IIb/IIIa, platelet-derived ß-thromboglobulin, interleukin-6, and interleukin-1ß compared to the free doxorubicin. Furthermore, 1000 µg/mL nanocomposite led to 0.2% hemolytic activity, much less than the 5% standard limit. The void nanocarrier induced no significant level of cytotoxicity in breast cancer and normal cells following 96 h incubation. The doxorubicin-loaded nanocomposite presented higher cytotoxicity, apoptosis induction, and doxorubicin uptake in cancer cells than free doxorubicin. Conversely, lower cytotoxicity, apoptosis induction, and doxorubicin uptake were observed in normal cells treated with the doxorubicin-loaded nanocarrier compared to free doxorubicin. In line with the results of in vitro experiments, in vivo studies on tumor-bearing mice showed more suppression of tumor growth by the doxorubicin-loaded nanocomposite compared to the free drug. Moreover, the pharmacokinetic study revealed slow release of doxorubicin from the nanocomposite. Besides, in vitro and in vivo MRI studies presented a higher r2/r1 ratio and comparable contrast to the commercially available DOTAREM, respectively. Our findings suggest that this new nanocomposite is a promising nanotheranostic system with promising potential for cancer therapy and diagnosis.

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

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.

Effect of glycated insulin on the blood-brain barrier permeability: An in vitro study.

Altered blood-brain barrier (BBB) permeability may contribute to pathogenesis of diabetes-related central nervous system disorders. Considering the presence of glycated insulin in plasma of type 2 diabetic patients, we hypothesized that glycated insulin could induce changes in paracellular permeability in BBB. Therefore, the authors decided to study the effect of glycated insulin on paracellular permeability in a BBB model and the change induced in insulin conformation upon glycation. In this study, the structural modification was examined by fluorescence and circular dichroism spectroscopies and dynamic light scattering. Cell proliferation and production of ROS in astrocytes and HUVEC cells were analyzed by MTT and spectrofluorometric assays, respectively. Apoptosis induction was determined and confirmed by flow cytometry and western blot analyses, respectively. The permeability was measured Lucifer yellow and FITC-Dextran. According to our results, glycated insulin presented altered conformation and more exposed hydrophobic patches than insulin. Formation of oligomeric species and advanced glycated end products (AGEs) were determined. Lower cell viability, higher apoptosis, and more ROS were detected upon treatment of cells with glycated insulin. Finally, glycated insulin led to increased Lucifer yellow and FITC-dextran transportation across the BBB model which could result from ROS producing and apoptosis-inducing activities of AGE-insulin.

Curcumin-lipoic acid conjugate as a promising anticancer agent on the surface of gold­iron oxide nanocomposites: A pH-sensitive targeted drug delivery system for brain cancer theranostics.

Brain tumor is a lethal, fast growing cancer and a difficult case for treatment. Receptor-mediated endocytosis has been recognized as one of the most effective methods for drug delivery to brain tissue by overcoming obstacles associated with conventional therapeutics. In this work, a targeted theranostic drug delivery system (DDS) was prepared based on gold­iron oxide nanocomposites (Fe3O4@Au NCs). Lipoic acid-curcumin (LA-CUR) was synthesized and introduced as a novel anticancer drug, and glutathione (GSH) was exploited as the targeting ligand. Both LA-CUR and GSH were easily attached to Fe3O4@Au NCs via Au-S interaction. As a negatively charged nanocarrier, the prepared DDS showed relatively less protein adsorption. Accordingly, hemocompatibility assays (complement, platelet, and leucocyte activation) revealed its hemocompatible virtue, especially in respect of free LA-CUR. GSH functionalization led to 2-fold increase of cellular uptake in GSH receptor-positive astrocyte cells which could primarily indicate the probable ability of the DDS to bypass BBB. Cytotoxicity and apoptosis assays together showed the noticeably enhanced cytotoxicity of LA-CUR against cancerous U87MG cells (IC50=2.69µg/ml) in comparison with curcumin (IC50=21.31µg/ml); moreover, the DDS demonstrated relatively higher cytotoxicity against cancerous U87MG cells than normal astrocyte cells which was in accordance with pH sensitive mechanism of LA-CUR release. Besides, the results of in vitro magnetic resonance imaging (MRI) (relaxation rate (r2)=80.73 (s-1·mM-1)) primarily revealed that the DDS can be applied as a negative MRI contrast agent. In sum, the prepared DDS appeared to be a promising candidate for brain cancer treatment and a favorable MRI contrast agent.

Photobiomodulation leads to enhanced radiosensitivity through induction of apoptosis and autophagy in human cervical cancer cells.

The radiomodulatory effect of photobiomodulation (PBM) has recently been studied in cancer cells. The aim of this study was to investigate cellular mechanisms involved in the X-ray radiosensitivity of HeLa cells pre-exposed to PBM. HeLa cells were irradiated with 685 nm laser at different energy densities prior to X-ray ionizing radiation. After irradiation, clonogenic cell survival, cell death due to apoptosis and autophagy were determined. Levels of intracellular reactive oxygen species (ROS), DNA damage and, cell cycle distribution after PBM were measured. PBM at different energy densities (5-20 J/cm2 ) was not cytotoxic. However, HeLa cells pre-exposed to 20 J/cm2 showed enhanced inhibition of colony formation following ionizing radiation. Enhanced radiosensitivity was due to increased oxidative stress, DNA damage, and radiation-induced apoptosis and autophagy. These results suggest that 685 nm PBM at a higher energy density could possibly be a promising radiosensitizing agent in cervical cancer, to decrease the radiation dose delivered, and therefore prevent the side-effects that are associated with cancer radiotherapy.

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.

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 MDA-MB-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, MDA-MB-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.

Antioxidant activity of low molecular weight alginate produced by thermal treatment.

By definition, antioxidants are molecules that inhibit the oxidation of other molecules. Therefore, such compounds have very important clinical roles. In this study alginate polymer was depolymerized by heat treatment. The resulting low molecular weight alginates were investigated by UV-visible spectroscopy, Viscometry, Dynamic light scattering and FT-IR spectroscopy techniques. Antioxidant properties of these heat products were studied by ABTS and superoxide radical scavenging assays. Results showed that heating caused breaks in the polymer chain and so generation of low molecular weight alginates. Antioxidant measurements confirmed antioxidant activity of alginate increased upon a decrease in molecular weight. Therefore, low molecular weight alginate produced by heating could be considered as a stronger antioxidant than alginate polymer. These products could be useful for industrial and biomedical applications.