FT-IR Spectral Signature of Sensitive and Multidrug-Resistant Osteosarcoma Cell-Derived Extracellular Nanovesicles

Osteosarcoma (OS) is the most common primary bone cancer in children and adolescents. Despite aggressive treatment regimens, the outcome is unsatisfactory, and multidrug resistance (MDR) is a pivotal process in OS treatment failure. OS-derived extracellular vesicles (EVs) promote drug resistance to chemotherapy and target therapy through different mechanisms. The aim of this study was to identify subpopulations of osteosarcoma-EVs by Fourier transform infrared spectroscopy (FT-IR) to define a specific spectral signature for sensitive and multidrug-resistant OS-derived EVs. EVs were isolated from sensitive and MDR OS cells as well as from mesenchymal stem cells by differential centrifugation and ultracentrifugation. EVs size, morphology and protein expression were characterized. FT-IR/ATR of EVs spectra were acquired in the region of 400-4000 cm(-1) (resolution 4 cm(-1), 128 scans). The FT-IR spectra obtained were consistently different in the EVs compared to cells from which they originate. A specific spectral signature, characterized by a shift and a new band (1601 cm(-1)), permitted to clearly distinguish EVs isolated by sensitive and multidrug-resistant OS cells. Our data suggest that FT-IR spectroscopy allows to characterize and define a specific spectral signature for sensitive and MDR OS-derived EVs

Sequential Combination of Cognitive-Behavioral Treatment and Well-Being Therapy in Depressed Patients with Acute Coronary Syndromes: A Randomized Controlled Trial (TREATED-ACS Study)

Introduction: Randomized controlled trials (RCT) of psychotherapeutic interventions have addressed depression and demoralization associated with acute coronary syndromes (ACS). The present trial introduces psychological well-being, an increasingly recognized factor in cardiovascular health, as a therapeutic target. Objective: This study was designed to determine whether the sequential combination of cognitive-behavioral therapy (CBT) and well-being therapy (WBT) may yield more favorable outcomes than an active control group (clinical management; CM) and to identify subgroups of patients at greater risk for cardiac negative outcomes. Methods: This multicenter RCT comparedCBT/WBT sequential combination versus CM, with up to 30 months of follow-up. One hundred consecutive depressed and/or demoralized patients (out of 740 initially screened by cardiologists after a first episode of ACS) were randomized to CBT/WBT associated with lifestyle suggestions (n = 50) and CM (n = 50). The main outcome measures included: severity of depressive symptoms according to the Clinical Interview for Depression, changes in subclinical psychological distress, well-being, and biomarkers, and medical complications and events. Results: CBT/WBT sequential combination was associated with a significant improvement in depressive symptoms compared to CM. In both groups, the benefits persisted at follow-up, even though the differences faded. Treatment was also related to a significant amelioration of biomarkers (platelet count, HDL, and D-dimer), whereas the 2 groups showed similar frequencies of adverse cardiac events. Conclusions: Addressing psychological well-being in the psychotherapeutic approach to ACS patients with depressive symptoms was found to entail important clinical benefits. It is argued that lifestyle changes geared toward cardiovascular health may be facilitated by a personalized approach that targets well-being

Blocking Tumor-Educated MSC Paracrine Activity Halts Osteosarcoma Progression

Purpose: Human osteosarcoma is a genetically heterogeneous bone malignancy with poor prognosis despite the employment of aggressive chemotherapy regimens. Because druggable driver mutations have not been established, dissecting the interactions between osteosarcoma cells and supporting stroma may provide insights into novel therapeutic targets.Experimental Design: By using a bioluminescent orthotopic xenograft mouse model of osteosarcoma, we evaluated the effect of tumor extracellular vesicle (EV)-educated mesenchymal stem cells (TEMSC) on osteosarcoma progression. Characterization and functional studies were designed to assess the mechanisms underlying MSC education. Independent series of tissue specimens were analyzed to corroborate the preclinical findings, and the composition of patient serum EVs was analyzed after isolation with size-exclusion chromatography.Results: We show that EVs secreted by highly malignant osteosarcoma cells selectively incorporate a membrane-associated form of TGF\u3b2, which induces proinflammatory IL6 production by MSCs. TEMSCs promote tumor growth, accompanied with intratumor STAT3 activation and lung metastasis formation, which was not observed with control MSCs. Importantly, intravenous administration of the anti-IL6 receptor antibody tocilizumab abrogated the tumor-promoting effects of TEMSCs. RNA-seq analysis of human osteosarcoma tissues revealed a distinct TGF\u3b2-induced prometastatic gene signature. Tissue microarray immunostaining indicated active STAT3 signaling in human osteosarcoma, consistent with the observations in TEMSC-treated mice. Finally, we isolated pure populations of EVs from serum and demonstrated that circulating levels of EV-associated TGF\u3b2 are increased in osteosarcoma patients.Conclusions: Collectively, our findings suggest that TEMSCs promote osteosarcoma progression and provide the basis for testing IL6- and TGF\u3b2-blocking agents as new therapeutic options for osteosarcoma patients. Clin Cancer Res; 23(14); 3721-33. \ua92017 AACR

AKT as a molecular target of fenretinide activity in glioblastoma in vitro

Glioblastoma multiforme is the most aggressive of the primary central nervous system tumours, with a median patient’s survival of less then 1 year. In this tumour, a constitutive activation of the serine/threonine protein kinase Akt/PKB pathway is associated with increase in tumour grade, decreased level of apoptosis and adverse clinical outcome. Fenretinide, a synthetic derivative of retinoic acid, is a modulator of cell proliferation and inductor of apoptosis in vitro; it is a very promising agent because of easy administration, long-term tolerability and low incidence of long-term side effects. It is currently under evaluation in clinical trials as a chemopreventive agent against a variety of cancers. Fenretinide has been shown to inhibit glioblastoma cells in vitro, but the mechanism of its antiproliferative action remains elusive. The present study was designed to investigate the role of Akt/PKB in the molecular mechanism of action of fenretinide in human glioblastoma in vitro, and for this purpose CRS-A2 and A-172 cell lines were chosen, which do highly express Akt/PKB. The dose- and time-dependent significance of cell survival inhibition was determined by Trypan Blue exclusion test. Apoptosis was checked by DNA fragmentation and caspase induction. Protein expression was evaluated by Western blotting analysis. Results show that the antiproliferative activity of Fenretinide in human glioblastoma in vitro, at pharmacologically achievable doses, is correlated with a downregulation of Akt protein expression as well as an inhibition of constitutively active Akt phosphorylation. In addition, the drug induced a down-regulation of cyclin D1/Cdk4 and a decrease of p21CIP1 protein expression.. These events preceded activation of caspases, proteolysis of the nuclear enzyme poly(ADP-ribose)polymerase (PARP) and DNA fragmentation in CRS-A2 glioma cells. No induction of apoptosis was evident in A-172 glioblastoma cells. Our data identified in the Akt/PKB pathway a new molecular target of fenretinide activity and provides a molecular rationale for therapeutic strategies in human glioblastomas

Multimodal transfer of MDR by exosomes in human osteosarcoma

Exosomes are extracellular vesicles released by both normal and tumour cells which are involved in a new intercellular communication pathway by delivering cargo (e.g., proteins, microRNAs, mRNAs) to recipient cells. Tumour-derived exosomes have been shown to play critical roles in different stages of tumour growth and progression. In this study, we investigated the potential role of exosomes to transfer the multidrug resistance (MDR) phenotype in human osteosarcoma cells. Exosomes were isolated by differential centrifugation of culture media from multidrug resistant human osteosarcoma MG-63DXR30 (Exo/DXR) and MG-63 parental cells (Exo/S). Exosome purity was examined by transmission electron microscopy and confirmed by immunoblot analysis for the expression of specific exosomal markers. Our data showed that exosomes derived from doxorubicin-resistant osteosarcoma cells could be taken up into secondary cells and induce a doxorubicin-resistant phenotype. The incubation of osteosarcoma cells with Exo/DXR decreased the sensitivity of parental cells to doxorubicin, while exposure with Exo/S was ineffective. In addition, we demonstrated that Exo/DXR expressed higher levels of MDR-1 mRNA and P-glycoprotein compared to Exo/S (p=0.03). Interestingly, both MDR-1 mRNA and P-gp increased in MG-63 cells after incubation with Exo/DXR, suggesting this as the main mechanism of exosome-mediated transfer of drug resistance. Our findings suggest that multidrug resistant osteosarcoma cells are able to spread their ability to resist the effects of doxorubicin treatment on sensitive cells by transferring exosomes carrying MDR-1 mRNA and its product P-glycoprotein

Multimodal transfer of MDR by exosomes in human osteosarcoma

Exosomes are extracellular vesicles released by both normal and tumour cells which are involved in a new intercellular communication pathway by delivering cargo (e.g., proteins, microRNAs, mRNAs) to recipient cells. Tumour-derived exosomes have been shown to play critical roles in different stages of tumour growth and progression. In this study, we investigated the potential role of exosomes to transfer the multidrug resistance (MDR) phenotype in human osteosarcoma cells. Exosomes were isolated by differential centrifugation of culture media from multidrug resistant human osteosarcoma MG-63DXR30 (Exo/DXR) and MG-63 parental cells (Exo/S). Exosome purity was examined by transmission electron microscopy and confirmed by immunoblot analysis for the expression of specific exosomal markers. Our data showed that exosomes derived from doxorubicin-resistant osteosarcoma cells could be taken up into secondary cells and induce a doxorubicin-resistant phenotype. The incubation of osteosarcoma cells with Exo/DXR decreased the sensitivity of parental cells to doxorubicin, while exposure with Exo/S was ineffective. In addition, we demonstrated that Exo/DXR expressed higher levels of MDR-1 mRNA and P-glycoprotein compared to Exo/S (p=0.03). Interestingly, both MDR-1 mRNA and P-gp increased in MG-63 cells after incubation with Exo/DXR, suggesting this as the main mechanism of exosome-mediated transfer of drug resistance. Our findings suggest that multidrug resistant osteosarcoma cells are able to spread their ability to resist the effects of doxorubicin treatment on sensitive cells by transferring exosomes carrying MDR-1 mRNA and its product P-glycoprotein

Assessing psychological factors affecting medical conditions: comparison between different proposals

OBJECTIVE: We compared the provisional Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5) criteria for Somatic Symptom Disorders (SSD) and an alternative classification based on the Diagnostic Criteria for Psychosomatic Research (DCPR) as to prevalence and associations with dimensional measures of psychological distress and functioning in a population of medical patients. METHOD: Seventy consecutive outpatients with congestive heart failure were administered an ad hoc structured clinical interview for the identification of DSM-5 SSD, the section concerning hypochondriasis of the Structured Clinical Interview for DSM-IV, the Structured Interview for DCPR and Paykel's Clinical Interview for Depression. Subjects also completed the Symptom Questionnaire and the Psychosocial Index. Global assessment of functioning was performed with the DSM-IV Axis V. RESULTS: A diagnosis within DSM-5 SSD was found in 13 patients (18.5%): 61.5% of them were diagnosed with the Psychological Factors Affecting Medical Condition category. Twenty-nine patients (41.4%) were classified according to the DCPR-based proposal: illness denial, demoralization and irritable mood were the most frequent specifiers. The DCPR-based classification showed a greater number of significant associations with dimensional measures of psychological distress, global functioning and stress. CONCLUSION: Compared to DSM-5 SSD, the DCPR-based proposal was more sensitive in detecting psychological factors relevant to illness course and provided a better characterization of such factors. The DCPR-based proposal was also superior in identifying patients with increased psychological distress and poor psychosocial functioning

Acridine Orange is an Effective Anti-Cancer Drug that Affects Mitochondrial Function in Osteosarcoma Cells

Acridine orange (AO) is an antimalarial drug that accumulates into acidic cellular compartments. Lysosomes are quite acidic in cancer cells, and on this basis we have demonstrated that photoactivated AO is selectively toxic in sarcomas. However, photodynamic therapy is only locally effective, and cannot be used to eradicate systemic residual disease. In this study, we have evaluated the activity of non-photoactivated AO on sensitive and chemoresistant osteosarcoma (OS) cells to be considered for the systemic delivery. Since lysosomes are even more acidic in chemoresistant cells (MDR), we found that AO accumulation was significantly higher in the lysosomes of MDR in respect to parental cells, and in both cell types, therapeutic doses of AO significantly inhibited cell growth. However, the level of growth inhibition was inversely related to the level of lysosomal uptake of AO, suggesting that the main target of this agent is indeed extralysosomal. A significant reduction of intracellular ATP content and of the expression of mitochondrial complex III suggests a mitochondrial targeting. Notably, MDR cells showed a lower mitochondrial activity. Finally, the combined treatment of AO with the anticancer agent doxorubicin (DXR) significantly increased chemotoxicity by promoting DXR mitochondrial targeting, as revealed by the further reduction in ATP intracellular content. In conclusion, AO is able to effectively target both sensitive and resistant OS cells through mitotoxicity

Exosome-like Nanovesicles Isolated from Citrus limon L. Exert Antioxidative Effect

Exosome-like nanovesicles are biological nanostructures mediating cell-tocell communication and capable to load selected cargos also in the interaction among different species