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Microparticles induce multifactorial resistance through oncogenic pathways independently of cancer cell type.

de Souza PS, Cruz AL, Viola JP, Maia RC - Cancer Sci. (2014)

Bottom Line: By co-culturing MP derived from MDR-positive cells with recipient cells, we showed that sensitive cells accumulated Pgp, IAP proteins and mRNA.In addition, MP promoted microRNA transfer and NFκB and Yb-1 activation.Therefore, our results indicate that MP can induce a multifactorial phenotype in sensitive cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Program of Hemato-Oncology Molecular, Brazilian National Cancer Institute, Rio de Janeiro, Brazil.

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NFκB and Yb-1 pathway profile are changed in multidrug resistance (MDR)-negative recipient cells after co-culturing with isolated membrane microparticles (MP). NFκB pathway was analyzed in A549 cells and MCF7 recipient cells through IκBα expression levels (a), NFκB subcellular localization by fluorescence microscopy (b), and cellular fractionation (c). Yb-1 pathway was analyzed in both recipient cells through total Yb-1 expression level (a), cellular fractionation (c) and subcellular localization by fluorescence microscopy (d). MDR-negative cells showed degradation of IκBα (a) and NFκB nuclear staining (b), confirmed by NFκB increase in cellular nuclear fractions after co-culturing with P-glycoprotein (Pgp)-positive MP (c). MDR-negative cells, after co-culturing with MDR-positive MP, showed no changes in total Yb-1 levels (a). MCF7 cells showed slight enhancement in nuclear fraction (c, right panel), confirmed by nuclear foci as indicated by arrows (d, lower panel). A549 cells showed a perinuclear staining as indicated by arrowheads (d, upper panel). β-actin was used as load control for total western blot, and lamin B and HSC70 were used as load and purity control of nuclear and cytoplasmatic fractionation, respectively. DAPI was used for nuclear staining (blue), along with polyclonal anti- NFκB staining (green) and polyclonal anti-Yb-1 staining (red). Images captured at 1000× magnification.
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fig05: NFκB and Yb-1 pathway profile are changed in multidrug resistance (MDR)-negative recipient cells after co-culturing with isolated membrane microparticles (MP). NFκB pathway was analyzed in A549 cells and MCF7 recipient cells through IκBα expression levels (a), NFκB subcellular localization by fluorescence microscopy (b), and cellular fractionation (c). Yb-1 pathway was analyzed in both recipient cells through total Yb-1 expression level (a), cellular fractionation (c) and subcellular localization by fluorescence microscopy (d). MDR-negative cells showed degradation of IκBα (a) and NFκB nuclear staining (b), confirmed by NFκB increase in cellular nuclear fractions after co-culturing with P-glycoprotein (Pgp)-positive MP (c). MDR-negative cells, after co-culturing with MDR-positive MP, showed no changes in total Yb-1 levels (a). MCF7 cells showed slight enhancement in nuclear fraction (c, right panel), confirmed by nuclear foci as indicated by arrows (d, lower panel). A549 cells showed a perinuclear staining as indicated by arrowheads (d, upper panel). β-actin was used as load control for total western blot, and lamin B and HSC70 were used as load and purity control of nuclear and cytoplasmatic fractionation, respectively. DAPI was used for nuclear staining (blue), along with polyclonal anti- NFκB staining (green) and polyclonal anti-Yb-1 staining (red). Images captured at 1000× magnification.

Mentions: Constitutive activation of the NFκB pathway has been observed in several solid and hematological neoplasms and has been associated with oncogenesis by controlling several key mechanisms, including anti-apoptotic and resistance gene regulation.23 Our results demonstrated that BIRC4 mRNA was not contained in MP derived from resistant cells (Fig.2b). However, we observed BIRC4 mRNA upregulation in both recipient cells after co-culturing with MDR-positive donor cells and MDR-positive cell-derived MP (Fig.3c and Suppl. Fig. S2, respectively). Thus, we evaluated NFκB pathway activation through IκBα regulation and NFκB subcellular localization. IκBα levels were reduced in A549 and MCF7 cells after co-culturing with MDR cell-derived MP (Fig.5a). Moreover, we observed a subcellular redistribution of NFκB with predominant nuclear localization after co-culturing (Fig.5b,c). These results strongly suggest the functional NFκB pathway involvement in intrinsic BIRC4 gene regulation. Likewise, nuclear NFκB may have contributed to intrinsic ABCB1 and IAP expression in recipient cells (Fig.3).


Microparticles induce multifactorial resistance through oncogenic pathways independently of cancer cell type.

de Souza PS, Cruz AL, Viola JP, Maia RC - Cancer Sci. (2014)

NFκB and Yb-1 pathway profile are changed in multidrug resistance (MDR)-negative recipient cells after co-culturing with isolated membrane microparticles (MP). NFκB pathway was analyzed in A549 cells and MCF7 recipient cells through IκBα expression levels (a), NFκB subcellular localization by fluorescence microscopy (b), and cellular fractionation (c). Yb-1 pathway was analyzed in both recipient cells through total Yb-1 expression level (a), cellular fractionation (c) and subcellular localization by fluorescence microscopy (d). MDR-negative cells showed degradation of IκBα (a) and NFκB nuclear staining (b), confirmed by NFκB increase in cellular nuclear fractions after co-culturing with P-glycoprotein (Pgp)-positive MP (c). MDR-negative cells, after co-culturing with MDR-positive MP, showed no changes in total Yb-1 levels (a). MCF7 cells showed slight enhancement in nuclear fraction (c, right panel), confirmed by nuclear foci as indicated by arrows (d, lower panel). A549 cells showed a perinuclear staining as indicated by arrowheads (d, upper panel). β-actin was used as load control for total western blot, and lamin B and HSC70 were used as load and purity control of nuclear and cytoplasmatic fractionation, respectively. DAPI was used for nuclear staining (blue), along with polyclonal anti- NFκB staining (green) and polyclonal anti-Yb-1 staining (red). Images captured at 1000× magnification.
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fig05: NFκB and Yb-1 pathway profile are changed in multidrug resistance (MDR)-negative recipient cells after co-culturing with isolated membrane microparticles (MP). NFκB pathway was analyzed in A549 cells and MCF7 recipient cells through IκBα expression levels (a), NFκB subcellular localization by fluorescence microscopy (b), and cellular fractionation (c). Yb-1 pathway was analyzed in both recipient cells through total Yb-1 expression level (a), cellular fractionation (c) and subcellular localization by fluorescence microscopy (d). MDR-negative cells showed degradation of IκBα (a) and NFκB nuclear staining (b), confirmed by NFκB increase in cellular nuclear fractions after co-culturing with P-glycoprotein (Pgp)-positive MP (c). MDR-negative cells, after co-culturing with MDR-positive MP, showed no changes in total Yb-1 levels (a). MCF7 cells showed slight enhancement in nuclear fraction (c, right panel), confirmed by nuclear foci as indicated by arrows (d, lower panel). A549 cells showed a perinuclear staining as indicated by arrowheads (d, upper panel). β-actin was used as load control for total western blot, and lamin B and HSC70 were used as load and purity control of nuclear and cytoplasmatic fractionation, respectively. DAPI was used for nuclear staining (blue), along with polyclonal anti- NFκB staining (green) and polyclonal anti-Yb-1 staining (red). Images captured at 1000× magnification.
Mentions: Constitutive activation of the NFκB pathway has been observed in several solid and hematological neoplasms and has been associated with oncogenesis by controlling several key mechanisms, including anti-apoptotic and resistance gene regulation.23 Our results demonstrated that BIRC4 mRNA was not contained in MP derived from resistant cells (Fig.2b). However, we observed BIRC4 mRNA upregulation in both recipient cells after co-culturing with MDR-positive donor cells and MDR-positive cell-derived MP (Fig.3c and Suppl. Fig. S2, respectively). Thus, we evaluated NFκB pathway activation through IκBα regulation and NFκB subcellular localization. IκBα levels were reduced in A549 and MCF7 cells after co-culturing with MDR cell-derived MP (Fig.5a). Moreover, we observed a subcellular redistribution of NFκB with predominant nuclear localization after co-culturing (Fig.5b,c). These results strongly suggest the functional NFκB pathway involvement in intrinsic BIRC4 gene regulation. Likewise, nuclear NFκB may have contributed to intrinsic ABCB1 and IAP expression in recipient cells (Fig.3).

Bottom Line: By co-culturing MP derived from MDR-positive cells with recipient cells, we showed that sensitive cells accumulated Pgp, IAP proteins and mRNA.In addition, MP promoted microRNA transfer and NFκB and Yb-1 activation.Therefore, our results indicate that MP can induce a multifactorial phenotype in sensitive cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Program of Hemato-Oncology Molecular, Brazilian National Cancer Institute, Rio de Janeiro, Brazil.

Show MeSH
Related in: MedlinePlus