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  • Please cite this article as Mei D

    2020-03-24

    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
    Figure 3 Active autophagy modulation of 7pep-M-RAP. (A) Quantitative analysis of cellular LC3B by ELISA assay (mean SD, n Z 3). **P < 0.01 vs Control; ,,P < 0.01 vs Free PTX; 66P < 0.01 vs 7pep-M-PTX. (B) Effects of different treatments with nanomedicines on the ratios of LC3-II to LC3-I. Levels of LC3B proteins expression were measured by western-blot, and the ratios of LC3-II to LC3-I were calculated by comparing the band densities (mean SD, n Z 3). *P < 0.05 vs Control. (C) Images and quantitative analysis of colocalization of the LC3B-labeled autophagic vesicles (green dots) and Lyso-tracker red-labeled lysosomes (red). The white scales represent 10 mm. Yellow-framed inserts show the scatter plots generated by Image J software (mean SD, n Z 6). *P < 0.05 vs Control; 66P < 0.01 vs 7pep-M-PTX. (D) Images and quantitative analysis of autophagic vacuoles specifically labeled by Cyto-ID dye (green). The cell nuclei were stained with Hoechst 33342 (blue). The white scales represent 10 mm. Each bar on the histogram represents mean fluorescence intensity obtained from 6 randomly selected DETA NONOate (mean SD, n Z 6).
    did not induce obvious apoptosis of MCF-7 cell, indicating the biocompatibility of micellar polymers. Both combination groups induced a higher proportion of apoptosis than either RAP or PTX preparations used alone, whereas the combination of 7pep-modi-fied nanocarriers (42.89%) prompted more apoptosis than non-modified micelles (31.00%). These observations were consistent with the results of cytotoxicity in vitro. Moreover, neither RAP formulations used alone induced obvious apoptosis, manifesting that RAP might not play a role of apoptosis inducer in the synergy effects of combination groups.
    3.4. Mechanism of the improved therapeutic efficacy of combination therapy
    3.4.1. Autophagy inhibitor 3-MA suppressed the cytotoxicity and autophagic vesicular accumulation of combination therapy Then, we explored the underlying mechanism for this synergy effect of 7pep-M-RAP combined with 7pep-M-PTX for breast cancer. Since 7pep-M-RAP could actively trigger autophagy as proved above, we assumed that excessive autophagy induced by high concentrations of RAP would cause damage to intracellular
    Table 2 IC50 values and CI50 values of RAP and PTX for different formulations used in single or in combination.
    e Not applicable.
    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 9
    Figure 4 Satisfactory synergistic chemotherapy with PTX. (A) Cytotoxicity of 7pep-M-Combi compared with 7pep-M-PTX or 7pep-M-RAP in single use (mean SD, n Z 6). *P < 0.05 and **P < 0.01, 7pep-M-PTX vs 7pep-M-Combi; #P < 0.05 and ##P < 0.01, 7pep-M-RAP vs 7pep-M-Combi. (B) IC50 values of RAP and PTX for different formulations used in single or in combination. (C) In vitro cell apoptosis evaluation of micelles against MCF-7 cells by flow cytometry. Double parameter dot plots show FITC-fluorescence (FL1-H axis) vs PI-fluorescence (FL2-H axis). Quadrants: lower left, normal live cells (annexin V /PI ); lower right, early apoptotic cell (annexin Vþ/PI ); upper right, late apoptotic or necrotic cells (annexin Vþ/PIþ); and upper left, mechanically injured cells (annexin V /PIþ).
    mitochondria or other organelles, and this effect, together with apoptosis induced by PTX, plays a role in promoting tumor cell death. In order to verify the above hypothesis, autophagy inhib-itor 3-MA was involved to evaluate the relevance between autophagy and anti-tumor effect. Furthermore, mitochondrial morphology, mitochondrial membrane potential, cytochrome C release, intracellular ATP content, and the activities of caspase-9 and 3 were investigated to examine the effect of co-administration induced autophagy on the morphology and func-tion of mitochondria.