Fig. 6.

Chemoresistant cells-derived exosomes enhance HIF1α binding to pGSN promoter region and induces CDDP resistance in chemosensitive OVCA cells. a Chemosensitive OVCA cells (target cells; OV4453, OV2295) were co-cultured with chemoresistant OVCA cells (OV90, OV866(2)), chemosensitive OVCA cells (OV2295), and pGSN-knocked down OV866(2) cells followed by CDDP treatment (10 µm; 24 h). Chemoresistant (OV90 and OV866(2)) but not the chemosensitive OVCA cells conferred CDDP resistance to chemosensitive OVCA cells. OV866(2) cells whose pGSN was knocked down failed to protect OV4453 and OV2295 against CDDP-induced apoptosis. b, c Conditioned media and exosomes from chemoresistant OVCA cells but not the chemosensitive cells increased pGSN content and conferred CDDP resistance to chemosensitive cells. A2780s cells were treated with conditioned media (B, 3 ml; 24 h) or exosomes (c–g, 40 µg/400,000 cells; 24 h) derived from cultures of A2780s, PA-1, Hey, OV2295, OV866(2), OV90, and A2780cp cells, and then cultured with or without CDDP (10 µM; 24 h). Exosomes were tagged with pCT-CD63-GFP (1 µg; 24 h) and their uptake by recipient cells (A2780s, labeled with PKH26 red fluorescent dyes) was assessed by IF. e, f Exosomes from chemoresistant cells depleted of pGSN failed to upregulate pGSN content and facilitated CDDP-induced apoptosis compared with exosomes with pGSN. Exosomal pGSN from chemoresistant OVCA cells confer resistance in OV2295 and OV4453 cells. g A2780s cells were cultured with exosomes (40 µg/400,000 cells; 24 h) derived from A2780s, A2780cp, and A2780cp following pGSN knockdown (A2780cp-pGSN-KD) after which they were treated with or without CDDP (10 µM; 24 h). pGSN and β-tubulin contents (loading control) were examined by WB. h HIF1α-pGSN promoter binding is higher in A2780cp than A2780s cells. A2780s and A2780cp cells were cultured with or without CDDP (10 µM; 24 h) and HIF1α-pGSN promoter binding was assessed by the CHIP assay. i Chemoresistant cells-derived exosomes increase HIF1α-pGSN promoter binding and attenuate CDDP-induced apoptosis in chemosensitive cells. A2780s cells were cultured with A2780cp cells-derived exosomes (40 µg/400,000 cells; 24 h), and then cultured with or without CDDP (10 µM; 24 h). HIF1α-pGSN promoter binding was assessed by ChIP assay. [a (a; ***p < 0.001 vs b); b (a; ***p < 0.001 vs b and c); d (a; ***p < 0.001 vs b and c); e (a; ***p < 0.001 vs b); f (a; ***p < 0.001 vs b); g (a; ***p < 0.001 vs b); h (a; **p < 0.01 vs b); i (a; **p < 0.01 vs b)]. N = 3