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  • br and function of mitochondria including TEM co localizatio

    2020-03-24


    and function of mitochondria, including TEM, co-localization of mitochondria with autophagic vesicles, change in MMP, release of cytochrome C, intracellular ATP levels, as well as activities of caspases 9 and 3.
    From TEM images in Fig. 6A and quantitative analysis results in Supporting Information Fig. S20, apoptotic body was observed in MCF-7 ATPγS tetralithium salt exposed to 7pep-M-PTX alone, confirming the presence of apoptotic cell death. In contrast, MCF-7 cells treated with 7pep-M-RAP alone or in combination with 7pep-M-PTX, contained a number of autophagic vacuoles, with digested mate-rials or dysfunctional mitochondria that were sequestered into a double-membrane-bound vesicle and lost their crista.
    Mitochondria were abnormal in appearance and an increased number of lysosomes were also observed. Consistent with the results obtained under an electron microscope, the confocal mi-croscopy revealed that the combination of 7pep-M-RAP and 7pep-M-PTX resulted in higher co-localization rate of Cyto-ID spe-cifically labeled-autophagic vesicles with Mitotracker labeled-mitochondria compared with monotherapy groups (Fig. 6B and Supporting Information Fig. S21). With the addition of 3-MA, both the accumulation of autophagic vesicles and co-localization of mitochondria with autophagic vesicles in combined therapy group were reduced and the number of mitochondria was increased. This might be due to the down-regulation of autophagy
    Please cite this article as: Mei D et al., Actively priming autophagic cell death with novel transferrin receptor-targeted nanomedicine for synergistic chemotherapy against breast cancer, Acta Pharmaceutica Sinica B, https://doi.org/10.1016/j.apsb.2019.03.006
    + MODEL
    Priming autophagic cell death with transferrin receptor-targeted nanomedicine 11
    Figure 6 Effect of co-administration induced autophagy on the morphology and function of mitochondria. (A) TEM micrographs of MCF-7 cells untreated (control) or treated with 7pep-M-PTX, 7pep-M-PTX, 7pep-M-Combi and 3-MA plus 7pep-M-Combi. Nucleus (yellow letter N), typical autophagosomes (red frames), damaged mitochondria (yellow frames) and apoptotic body (yellow arrows) are indicated. The yellow scales represent 1.0 mm. (B) Images and quantitative analysis of colocalization (yellow dots) of Cyto-ID specifically labeled autophagic vesicles (green dots) and Mitotracker labeled mitochondria (red) in MCF-7 cells treated without or with 3-MA. The white scales represent 25 mm. (C) Decline of mitochondria membrane potential (Dj) measured by flow cytometry. Live MCF-7 cells were stained with JC-10 after exposed to various for-mulations. (D) Quantitative analysis of cellular cytochrome C by ELISA assay (mean SD, n Z 3).^^P < 0.01 vs Control; * *P < 0.01 vs 7pep-M-PTX without 3-MA; #P < 0.05 vs 7pep-M-Combi without 3-MA. (E) Quantitative analysis of cellular ATP measured by luminometer (mean SD, n Z 3).^^P < 0.01 vs Control; **P < 0.01 vs 7pep-M-PTX without 3-MA; #P < 0.05 vs 7pep-M-PTX with 3-MA. (F) Activity of caspase-9 in MCF-7 cells induced by various formulations (mean SD, n Z 3).^^P < 0.01 vs Control; **P < 0.01 vs 7pep-M-PTX without 3-MA; #P < 0.05 vs 7pep-M-Combi without 3-MA.
    by 3-MA and the reduction of mitophagy. Selective degradation of mitochondria by autophagy is also known as ‘mitophagy’ and is considered to be promoted by their functional impairment and/or by MMP. Mitophagy may ensure the removal of damaged and potentially dangerous mitochondria, while excessive autophagy
    may cause damage to the function of organelles39e41. Since decline in MMP is a morphological characteristic of mitochon-drial function recession, we then used JC-10 to detect the changes in MMP by flow cytometry and confocal analysis. JC-10 is a cationic lipophilic fluorescent dye. It accumulates in mitochondria