Metformin Increases Exosome Biogenesis and Secretion in U87 MG Human Glioblastoma Cells: A Possible Mechanism of Therapeutic Resistance.

BACKGROUND: Glioblastoma multiforme (GBM) is the most common and aggressive brain tumor. Metformin, an anti-diabetic drug, can suppress tumor cells. Exosomes from GBM cells contribute to intercellular communication, tumor aggressiveness, and therapeutic resistance. We studied the effect of metformin on the exosomal secretory pathway in U87 MG cells. METHODS: Cell survival against metformin was investigated using MTT assay. Expression of miRNA-21, miRNA-155, and miRNA-182, as well as the genes involved in exosome biogenesis and secretion such as Rab27a, Rab27b, Rab11, CD63, and Alix were calculated by real time-PCR. The expression of CD63 protein was analyzed by western blotting, while the subcellular distribution of CD63 protein was monitored by flow cytometry. Exosomes were characterized by transmission and scanning electron microscopes, and flow cytometry. Amount of exosomes was assayed using acetylcholinesterase activity assay and ELISA. The expression of autophagic markers LC3 and P62 were assessed using ELISA. RESULTS: Data showed that metformin decreased cell survival and expression of miRNA-21, miRNA-155, and miRNA-182 (p <0.05). Expression of Rab27a, Rab27b, Rab11, CD63, and Alix as well as protein level of CD63 up-regulated in treated cells (p <0.05). Concurrently, flow cytometry analysis showed that surface CD63/total CD63 ratio was increased in treated cells (p <0.05). We found that acetylcholinesterase activity and CD63 protein of exosomes from treated cells increased (p <0.05). The expression of LC3 and P62 was not affected by metformin (p >0.05). CONCLUSION: Data indicates metformin could promote exosome biogenesis and secretion in U87 MG cells, proposing the therapeutic response against metformin.

Encapsulation of Leflunomide (LFD) in a novel niosomal formulation facilitated its delivery to THP-1 monocytic cells and enhanced Aryl hydrocarbon receptor (AhR) nuclear translocation and activation.

BACKGROUND: Leflunomide (LFD) is an Aryl hydrocarbon receptor (AhR) agonist and immunomodulatory drug with several side effects. Niosomes are novel drug delivery systems used to reduce the unfavorable effects of drugs by enhancing their bioavailability, controlling their release and targeting specific sites. OBJECTIVES: Here, we prepared niosomal formulations of LFD, evaluated their properties and delivered to THP-1 monocytic cells to study the activation and nuclear translocation of AhR. METHODS: Four types of non-ionic surfactants were utilized to formulate niosomes by thin film hydration (TFH) method. Entrapment efficiency (EE %) of niosomes were quantified and dynamic light scattering (DLS) was performed. Transmission electron microscopy (TEM) was used to identify the morphology of LFD niosomes. Dialysis method was used to measure LFD release rate. MTS assay was adopted to examine the viability of the cells upon each treatment. The nuclear transfer of AhR was investigated by Immunocytochemistry (ICC). The mRNA expression of IL1ß and CYP1A1 were evaluated using quantitative RT-PCR. RESULTS: Span 60: cholesterol (1:1) showed the highest EE% (70.00 ± 6.24), largest particles (419.00 ± 4.16 nm) and the best uniformity with the lowest PDI (0.291 ± 0.007). TEM micrographs of Span 60 (1:1) nanoparticles showed conventional spherical vesicles with internal aqueous spaces. The release rate of LFD from Span 60 (1:1) vesicles was slower. Although the viability of LFD niosome-treated THP-1 cells was decreased, they were associated with lower cytotoxic effects compared with the free LFD counterparts. Both free and niosomal LFD treatments intensified the nuclear translocation of AhR. The mRNA expression of CYP1A1 was overexpressed while IL1ß was downregulated in both free and niosomal LFD treated combinations. CONCLUSION: LFD encapsulation in Span 60: cholesterol (1:1) niosomal formulation could be introduced as a suitable vehicle of transferring LFD to THP-1 cells, with minimal cytotoxic effects, enhancing the AhR nuclear translocation and activation and inducing immunomodulatory properties. Graphical abstract The Graphical abstract; it demonstrates the workflow of the study and summary of results in brief.

Tumor-derived extracellular vesicles: reliable tools for Cancer diagnosis and clinical applications.

BACKGROUND: Studies have recently revealed that almost every type of cells including tumor cells abundantly release small vesicles known as extracellular vesicles (EVs) into the extracellular milieu. EVs carry a repertoire of biological molecules including nucleic acids, proteins, lipids, and carbohydrates and transport their cargo between cells in the vicinity as well as distantly located cells and hence act as messengers of intercellular communication. In this review, we aimed to discuss the tumor-derived exosome biology and the pivotal roles of exosomes in cancer diagnosis and treatment. METHODS: In the present review study, the authors studied several articles over the past two decades published on the kinetics of EVs in tumor environment as well as on the application of these vesicles in cancer diagnosis and therapy. RESULTS: A growing body of evidence indicates that nucleic acids such as microRNAs (miRNAs) transferring by EVs participate to create a conducive tumor environment. As EV-associated miRNAs are tissue-specific and present in most biological fluids, they hold great potential for clinical application in cancer early diagnosis, prognosis, and treatment response. Furthermore, exosomes can serve as drug delivery vehicles transferring miRNAs as well as therapeutic agents to target cells. These nano-vesicles exhibit ideal properties in comparison with the synthetic carriers that attracted scientist's attention in the field of nanotechnology medicine. Scientists have employed different strategies to build exosomes-based drug delivery system. In general, two methods (direct engineering and indirect engineering) are being utilized to produce artificial exosomes. Para-clinical data have confirmed the beneficial effects of engineering exosomes in cancer therapy. CONCLUSION: Exosomal miRNAs hold great promise for clinical application in early diagnosis and treatment of cancers. In addition, in spite of enthusiastic results obtained by engineered exosomes, however, there is an increasing concern over the use of optimal methods for engineering exosomes and the safety of engineered exosomes in clinical trials is still unclear.