Detection of TNF-α Protein in Extracellular Vesicles Derived from Tumor Cells by Western Blotting.

Detection of tumor necrosis factor-alpha (TNF-α) is usually performed in cell cultured medium or body fluids via measurement of its soluble extracellular form. However, depending on cellular condition, TNF-α might be transported through extracellular vesicles (EV) from donor cells to recipient cells. EV are small membrane-delimited structures (∼50 nm to 10 µm) that are spontaneously released from multiple cell types. In cancer, EV arise as important mediators in intercellular communication, and their molecular content may support tumor progression. This chapter describes methods to identify protein content in EV released from the tumor cell cultures. Through this protocol, we show first how to purify EV from in vitro cell culture by using differential centrifugation technique and then we demonstrate how to identify both membrane and soluble TNF-α forms in EV by Western blotting.

TNF-α Modulates P-Glycoprotein Expression and Contributes to Cellular Proliferation via Extracellular Vesicles.

P-glycoprotein (Pgp/ABCB1) overexpression is associated with multidrug resistance (MDR) phenotype and, consequently, failure in cancer chemotherapy. However, molecules involved in cell death deregulation may also support MDR. Tumor necrosis factor-alpha (TNF-α) is an important cytokine that may trigger either death or tumor growth. Here, we examined the role of cancer cells in self-maintenance and promotion of cellular malignancy through the transport of Pgp and TNF-α molecules by extracellular vesicles (membrane microparticles (MP)). By using a classical MDR model in vitro, we identified a positive correlation between endogenous TNF-α and Pgp, which possibly favored a non-cytotoxic effect of recombinant TNF-α (rTNF-α). We also found a positive feedback involving rTNF-α incubation and TNF-α regulation. On the other hand, rTNF-α induced a reduction in Pgp expression levels and contributed to a reduced Pgp efflux function. Our results also showed that parental and MDR cells spontaneously released MP containing endogenous TNF-α and Pgp. However, these MP were unable to transfer their content to non-cancer recipient cells. Nevertheless, MP released from parental and MDR cells elevated the proliferation index of non-tumor cells. Collectively, our results suggest that Pgp and endogenous TNF-α positively regulate cancer cell malignancy and contribute to changes in normal cell behavior through MP.

Characterization of a multidrug-resistant chronic myeloid leukemia cell line presenting multiple resistance mechanisms.

The multidrug-resistant (MDR) phenotype is multifactorial, and cell lines presenting multiple resistance mechanisms might be good models to understand the importance of the various pathways involved. The present work characterized a MDR chronic myeloid leukemia cell line, derived from K562 through a selective process using daunorubicin. This MDR cell line was shown to be resistant to vincristine, daunorubicin, and partially resistant to imatinib. It showed a slower duplication rate. Overexpression of ABCB1 and ABCC1 was observed at the protein and functional levels and the expression of CD95, a molecule related to cell death, was reduced in the MDR cell line. Conversely, no differences were observed related to the anti-apoptotic molecule Bcl-2 or p53 expression. The activation antigen CD69 was reduced in the MDR cell line and treatment with imatinib further decreased the expressed levels. Furthermore, secretion of IL-8 was diminished in the MDR cell line. When daunorubicin-selected cells were compared to another MDR cell line, Lucena 1, derived from the same parental line K562, and selected with vincristine, a different profile was observed in relation to most aspects studied. When both cell lines were silenced for ABCB1, differences in CD69 and CD95 were maintained, despite resistance reversal. These results reinforce the idea that cell lines selected in vitro may display multiple resistance strategies that may vary with the selective agent used as well as during different steps of the selection process.