br CI Z IC PTX Combi
CI50 ZðIC50PTXÞCombi þ
ðIC50RAPÞCombi ð 1 Þ
The index of CI50 was used to assess the synergetic, additive or antagonistic effect of various PTX used in combination with RAP formulations30.
2.5. Cell apoptosis analysis by flow cytometry
Cell apoptosis was evaluated by Annexin V-FITC/PI apoptosis detection kit. Briefly, MCF-7 KIN59 were cultured in 6-well plates for 24 h, and treated with culture medium (negative control), free RAP, M-RAP, 7pep-M-RAP, free PTX, M-PTX, 7pep-M-PTX, Free Combi, M-Combi, 7pep-M-Combi for 24 h. For combination therapy, RAP formulations were added 12 h before the treatment of PTX formulations. The final concentrations of RAP and PTX were 100 and 10 nmol/L, respectively. At the end of treatment, cells were trypsinized, washed with cold PBS. Then cells were stained with Annexin V-FITC, followed by propidium iodide (PI). Finally, the cell apoptosis was analyzed by a flow cytometer with 10,000 events collected.
2.6. The mechanism study on the synergetic effect of combination therapy
2.6.1. The effect of autophagy inhibitor 3-MA on cytotoxicity and apoptosis
MCF-7 cells were cultured for 24 h at 37 C. Then cells were treated with or without 3-MA for 6 h, followed by 7pep-M-RAP, 7pep-M-PTX, 7pep-M-Combi, respectively. For combination group, 7pep-M-PTX was added 12 h after 7pep-M-RAP. The final concentrations of RAP and PTX were 100 nmol/L and 10 nmol/L, respectively. After the addition of 7pep-M-PTX for 24 h, the cells
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
were treated with SRB colorimetric assay, or analyzed by annexin V-FITC/PI apoptosis detection kit.
2.6.2. The inhibition effect of 3-MA on autophagic vesicular accumulation
The cellular autophagic vesicular accumulation in different treatment groups was determined by monodansylcadaverine (MDC) staining, Cyto-ID autophagy detection kit, transmission electron microscopy (TEM), the expression of the autophagy marker protein LC3B, as well as the LC3-II/LC3-I ratio detected by Western blot. MCF-7 cells were seeded on coverslips for 24 h and treated with different RAP or PTX formulation used in single or combination as described above.
22.214.171.124. MDC labeling. Drugs treated cells were stained with 10 mmol/L MDC at 37 C for 15 min31. The cellular fluorescence was observed using fluorescence microscope (Olympus, FV1000, Tokyo, Japan).
126.96.36.199. Cyto-ID autophagy detection dye staining. After treated with drugs for 24 h, cells were stained with Cyto-ID autophagy detection dye plus Hoechst 33342 at 37 C for 30 min. Stained cells were then photographed with a CLSM, or analyzed by flow cytometer. The excitation and emission wavelengths for Cyto-ID dye were 488 and 560 nm, respectively.
188.8.131.52. ELISA analysis and immunofluorescent staining of LC3B. Briefly, for ELISA analysis, after treated with drugs for 24 h, cells were lysed, centrifuged at 1500 g at 4 C for 5 min. Samples containing LC3B were incubated in the wells of micro-plate pre-processed by antibody for 2 h. After being washed, the microplate was incubated with HRP-conjugate reagent for another 1 h. The substrates were added for color development in the dark for 30 min. The absorbance was measured at 450 nm and the expression ratio was calculated according to the user manual. For immunofluorescence staining, after treated with drugs, cells were fixed, incubated with anti-LC3B monoclonal antibody overnight at 4 C, followed by staining with second antibody (Texas red labeled goat IgG). Negative control cells were incubated with 5% BSA solution instead of anti-LC3B antibody. Nuclei were labeled with Hoechst 33258 for 20 min at 37 C. Finally, cells were observed using a CLSM.