br As depicted in Fig A and B as
As depicted in Fig. 5A and B, as well as Supporting Information Figs. S17 and S18, the cell viability of 7pep-M-PTX monotherapy, combination group, and combination therapy plus 3-MA were 50.53 4.04%, 30.67 4.61%, 40.54 4.13%, respectively, and the apoptosis proportion in above three treatment groups were 15.5 4.14%, 45.94 3.33%, 37.12 4.84%, respectively. The results of SRB cytotoxicity assay and annexin V-FITC/PI apoptosis analysis consistently demonstrated that the combination use of 7pep-M-RAP and 7pep-M-PTX enhanced the chemosensitivity to 7pep-M-PTX in TfR-overexpressing breast cancer cells, and this synergistic interaction were significantly suppressed by autophagy inhibitor 3-MA (P < 0.05), suggesting that 7pep-M-RAP-induced autophagy might play an active role in enhanced anti-tumor effect.
Cyto-ID autophagy detection and LC3B protein aggregation determination were also applied to further confirm whether the synergism induced by the combination treatment involved the active autophagy modulation effect of 7pep-M-RAP. Qualitative and quantitative analysis of Cyto-ID staining further verified that 7pep-M-RAP alone or in combination with 7pep-M-PTX could induce similar levels of autophagic vesicle accumulation, while 3-MA could effectively inhibit the autophagic vacuoles
accumulation (Fig. 5C and D). Moreover, with the addition of 3-MA, the ratios of LC3-II to LC3-I expression, fluorescence dots of LC3B aggregation, and the quantity of LC3B measured by ELISA consistently decreased significantly both in single 7pep-M-RAP and combination group (Fig. 5E and F, and Supporting Information Fig. S19).
Therefore, we came to a preliminary conclusion that the autophagy induced by 7pep-M-RAP played an important role in the synergistic effect of combination therapy. It is worth noting that 3-MA could not completely reverse the efficacy of co-administration, indicating that autophagy is one of the many mechanisms for the synergy between 7pep-M-RAP and 7pep-M-PTX. The currently reported synergistic anti-tumor mechanisms of combined RAP (or its derivatives) with PTX mainly involves the
anti-angiogenesis effect of RAP and its direct inhibition of mTOR in tumor development11,13,35. It was reported that PTX down-
regulates Akt Epoprostenol followed by mTOR activation, which promotes resistance to chemotherapy and hormone thera-pies in breast cancer cells, while RAP blocks the phosphoinositide 3-kinase (PI3K)/AKT/mTOR/p70S6K pathway, thus reversing drug resistance36e38. Besides, the inhibition of mTOR arrests the cells cycle in G1 phase, while PTX leads to G2/M phase arrest, this dual role leads to synergy effects15. However, the relationship between autophagy and synergistic effect of this combined strat-egy is not well understood. Therefore, our research attempted to clarify the role of RAP-induced autophagy in the combined anti-tumor efficacy.
3.4.2. Effect of co-administration induced autophagy on the morphology and function of mitochondria
To further assess the effect of co-administration induced auto-phagy on intracellular organelles, we examined the morphology
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
Figure 5 The inhibition effect of autophagy inhibitor 3-MA on cytotoxicity, apoptosis and intracellular autophagic vesicular accumulation. (A) The effect of 3-MA on the cytotoxicity induced by 7pep-M-PTX in single or in combination with 7pep-M-RAP (mean SD, n Z 6). 66P < 0.01 vs control without 3-MA; ##P < 0.01 vs 7pep-M-PTX without 3-MA; *P < 0.05 vs 7pep-M-Combi without 3-MA;^P < 0.05 vs 7pep-M-PTX without 3-MA. (B) Quantitative analysis of in vitro cell apoptosis based on flow cytometric plots (mean SD, n Z 4). Each bar represents the sum of early apoptotic cells and late apoptotic cells. **P < 0.01 vs 7pep-M-PTX without 3-MA; #P < 0.05 vs 7pep-M-Combi without 3-MA;^^P < 0.01 vs 7pep-M-PTX without 3-MA. (C) The effect of 3-MA on the Cyto-ID specifically labeled autophagic vesicles detected by flow cytometry. (D) The inhibition effect of 3-MA on the Cyto-ID dye specifically labeled autophagic compartments. Images showed the colocalization of the Cyto-ID fluorescence dye (green) and Hoechst 33342 (blue). The white scales represent 25 mm. (E) Levels of LC3-II to LC3-I 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 < 0.01 vs Control. ##P < 0.01 vs 7pep-M Combi without 3-MA. (F) The inhibition effect of 3-MA on the LC3B labeled autophagic vesicles. The white scale bars represent 75 mm.