br Please cite this article as Mei D et
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
and its intake is proportional to the MMP. As indicated in Fig. 6C and Supporting Information Fig. S22, the fluorescence intensity of JC-10 was obviously lower in the Echinomycin exposed to 7pep-M-RAP in combination with 7pep-M-PTX relative to the other groups, while the fluorescence intensity increased with the pretreatment of 3-MA, suggesting that the decrease of MMP induced by co-administration might be related to autophagy.
Fig. 6D and F, as well as Supporting Information Fig. S23 present the change in function of mitochondria with different treatments. It was showed that when compared with monotherapy, 7pep-M-RAP plus 7pep-M-PTX induced increased release of cytochrome c from mitochondria (Fig. 6D), decreased intracellular ATP content (Fig. 6E), as well as enhanced activities of caspase-9 and -3 (Fig. 6F and Supporting Information Fig. S23), and these effects could be partly suppressed by 3-MA. These findings showed that the mitochondrial morphology and function were impaired by the combination therapy, and this effect on mito-chondria might be partially associated with 7pep-M-RAP-induced autophagy.
Mitochondria are vital organelles for cellular metabolism and bioenergetics of eukaryotic cells, involving a variety of biological processes, such as intracellular homeostasis, cell proliferation, senescence, and death42. Based on the above results, it could be speculated that during the combination therapy of 7pep-M-RAP and 7pep-M-PTX, mitophagy occurred in MCF-7 cells, which led to mitochondrial damage, followed by mitochondrial membrane permeability transition, oxidative phosphorylation decoupling, excessive depletion of ATP, and the release of cytochrome c into cytoplasm after mitochondrial swelling43. After cytochrome c release, caspase 9 was activated by a series of signaling pathways that activated downstream cascade enzymes, such as caspase-3, causing a downstream cascade that eventually triggered apoptosis44. In short, it was demonstrated that 7pep-M-RAP
enhanced 7pep-M-PTX-induced mitochondria-associated apoptosis at least partly through autophagy activation, thereby enhancing the anti-tumor effect of 7pep-M-PTX.
Studies have shown that RAP at a concentration of greater than 100 nmol/L brought on autophagy-related death in drug-resistant NIH 3T3 cells45, which is similar to our results on MCF-7 cells in this study. It could be deduced that when 7pep-M-RAP is administered simultaneously with 7pep-M-PTX, or 7pep-M-RAP is administered after 7pep-M-PTX, cytotoxic PTX would firstly cause organelle damage, and then the autophagy induced by RAP might play a role in protecting MCF7 breast cancer cells from organelle damage caused by PTX. On the contrary, the pretreat-ment with 7pep-M-RAP on MCF-7 cells induced a large number of autophagy, resulting in excessive depletion of key intracellular processes and rapid accumulation of intracellular autophagic vesicles, whereas cells could not degrade the contents in time, leading to abnormal accumulation of autophagosomes and imbalance of cell functions46,47. During autophagy development, the non-specific capture of cytoplasmic components led to irre-versible damage to mitochondria or other organelles. At this time, the application of cytotoxic chemotherapeutic agents, such as PTX, aggravated the injury of organelles, which eventually led to cell death. Under the circumstances, the intracellular balance was broken, and autophagy no longer played a protective role, but promoted cell death.
In summary, based on our findings, it can be concluded that a combined strategy targeting autophagic cell death has great poten-tial for enhancing the antitumor efficacy of chemotherapeutic agents. However, in view of the biphasic effect of autophagy, there is