Exosomes, small (40–100 nm) extracellular membranous vesicles, attract enormous research interest because they are carriers of disease markers and a prospective delivery system for therapeutic agents. Differential centrifugation, the prevalent method of exosome isolation, frequently produces dissimilar and improper results because of the faulty practice of using a common centrifugation protocol with different rotors. Moreover, as recommended by suppliers, adjusting the centrifugation duration according to rotor K-factors does not work for “fixed-angle” rotors. For both types of rotors – “swinging bucket” and “fixed-angle” – we express the theoretically expected proportion of pelleted vesicles of a given size and the “cut-off” size of completely sedimented vesicles as dependent on the centrifugation force and duration and the sedimentation path-lengths. The proper centrifugation conditions can be selected using relatively simple theoretical estimates of the “cut-off” sizes of vesicles. Experimental verification on exosomes isolated from HT29 cell culture supernatant confirmed the main theoretical statements. Measured by the nanoparticle tracking analysis (NTA) technique, the concentration and size distribution of the vesicles after centrifugation agree with those theoretically expected. To simplify this “cut-off”-size-based adjustment of centrifugation protocol for any rotor, we developed a web-calculator.

Metformin, a biguanide antidiabetic drug, is used to decrease hyperglycemia in patients with type 2 diabetes. Recently, the epidemiological studies revealed the potential of metformin as an anti-tumor drug for several types of cancer, including breast cancer. Anti-tumor metformin action was found to be mediated, at least in part, via activation of adenosine monophosphate-activated protein kinase (AMPK)-intracellular energy sensor, which inhibits the mammalian target of rapamycin (mTOR) and some other signaling pathways. Nevertheless, some patients can be non-sensitive or resistant to metformin action. Here we analyzed the mechanism of the formation of metformin-resistant phenotype in breast cancer cells and its role in estrogen receptor (ER) regulation. The experiments were performed on the ER-positive MCF-7 breast cancer cells and metformin-resistant MCF-7 subline (MCF-7/M) developed due to long-term metformin treatment. The transcriptional activity of NF-jB and ER was measured by the luciferase reporter gene analysis. The protein expression was determined by immunoblotting (Snail1, (phospho)AMPK, (phospho)IjBa, (phospho)mTOR, cyclin D1, (phospho)Akt and ERa) and immunohistochemical analysis (E-cadherin). We have found that: 1) metformin treatment of MCF-7 cells is accompanied with the stimulation of AMPK and inhibition of growth-related proteins including IjBa, NF-jB, cyclin D1 and ERa; 2) long-term metformin treatment lead to the appearance and progression of cross-resistance to metformin and tamoxifen; the resistant cells are characterized with the unaffected AMPK activity, but the irreversible ER suppression and constitutive activation of Akt/Snail1 signaling; 3) Akt/Snail1 signaling is involved into progression of metformin resistance. The results presented may be considered as the first evidence of the progression of cross-resistance to metformin and Abbreviations: AMPK, adenosine monophosphate-activated protein kinase; EMT, epithelial-mesenchymal transition; ER, estrogen receptor; ERE, estrogen-responsive elements; mTOR, mammalian target of rapamycin 281tamoxifen in breast cancer cells. Importantly, the acquired resistance to both drugs is based on the constitutive activation of Akt/Snail1/E-cadherin signaling that opens new perspectives to overcome the metformin/tamoxifen resistance of breast cancer. V C 2016 IUBMB Life, 68(4): [281][282][283][284][285][286][287][288][289][290][291][292] 2016

Exosomes are small vesicles which are produced by the cells and released into the surrounding space. They can transfer biomolecules into recipient cells. The main goal of the work was to study the exosome involvement in the cell transfer of hormonal resistance. The experiments were performed on in vitro cultured estrogen-dependent MCF-7 breast cancer cells and MCF-7 sublines resistant to SERM tamoxifen and/or biguanide metformin, which exerts its anti-proliferative effect, at least in a part, via the suppression of estrogen machinery. The exosomes were purified by differential ultracentrifugation, cell response to tamoxifen was determined by MTT test, and the level and activity of signaling proteins were determined by Western blot and reporter analysis. We found that the treatment of the parent MCF-7 cells with exosomes from the resistant cells within 14 days lead to the partial resistance of the MCF-7 cells to antiestrogen drugs. The primary resistant cells and the cells with the exosome-induced resistance were characterized with these common features: decrease in ERα activity and parallel activation of Akt and AP-1, NF-κB, and SNAIL1 transcriptional factors. In general, we evaluate the established results as the evidence of the possible exosome involvement in the transferring of the hormone/metformin resistance in breast cancer cells.

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The purpose of this study was to identify critical pathways promoting survival of tamoxifen-tolerant, estrogen receptor  positive (ER+) breast cancer cells, which contribute to therapy resistance and disease recurrence. Gene expression profiling and pathway analysis was performed in ER+ breast tumors of patients before and after neo-adjuvant tamoxifen treatment and demonstrated activation of the NFκB pathway and an enrichment of EMT/stemness features.Exposure of ER+ breast cancer cell lines to tamoxifen, in vitro and in vivo, gives rise to a tamoxifen-tolerant population with similar NFκB activity and EMT/stemness characteristics.Small molecule inhibitors and CRISPR/Cas9 knock out were used to assess the role of the nuclear factor κB (NFκB) pathway and demonstrated that survival of tamoxifen-tolerant cells requires NFκB activity. Moreover, this pathway was essential for tumor recurrence following tamoxifen withdrawal. These findings establish that elevated NFκB activity is observed in breast cancer cell lines under selective pressure with tamoxifen in vitro and in vivo, as well as in patient tumors treated with neo-adjuvant tamoxifen therapy. This pathway is essential for survival and regrowth of tamoxifen-tolerant cells, and, as such, NFκB inhibition offers a promising approach to prevent recurrence of ER+ tumors following tamoxifen exposure.Implications: Understanding initial changes that enable survival of tamoxifen-tolerant cells, as mediated by NFκB pathway, may translate into therapeutic interventions to prevent resistance and relapse, which remain major causes of breast cancer lethality.