The manganese(III) porphyrin MnTnHex-2-PyP5+ modulates intracellular ROS and breast cancer cell migration: Impact on doxorubicin-treated cells

Fig. 1. Chemical structure of MnTnHex-2-PyP5+[9].

Fig. 2. Treatment with MnP and low concentrations of dox does not induce cell death. MCF7 (A, C and E) and MDA-MB-231 (B, D and F) cell viability and cell death induction following exposure to the indicated MnP and dox concentrations, were evaluated by an MTT assay (A and B) and a DNA content assay after cell fixation (C-F), respectively. Histograms show representative MCF7 and MDA-MB-231 DNA content profiles following exposure to dox (0.1 μM), MnP (5 μM) or both, fixation and PI stain (C–D). Summary results from cell viability (A and B) and DNA content assays (E and F) are represented as means ± SD. PI, propidium iodide.

Fig. 3. MnP and dox lead to an increase in intracellular ROS. Intracellular ROS levels were determined in MCF7 (A, B, C and E) or MDA-MB-231 (D and F) cells treated with the indicated drugs (dox (0.1 μM), MnP (5 μM)). Fluorescence microscopy images show representative MCF7 cells after 25-min incubation with DHR and DHE (A and B). Scale bars = 20 µm. Summary results (means ± SD from at least three independent experiments) show relative DHR and DHE fluorescence (C to F). *p < 0.05, **p < 0.01, ***p < 0.001 (one-way ANOVA with Tukey's test, relative to untreated cells).

Fig. 4. MnP and dox can reduce chemotaxis and random and collective cell migration. Collective cell migration, chemotaxis and random migration of MCF7 (A, C and F) or MDA-MB-231 (B, D and G) cells treated with the indicated drugs (dox (0.1 μM), MnP (5 μM)) were measured. Collective cell migration was measured by the wound healing assay (A and B), chemotaxis was measured using a transwell system with FBS as chemoattractant (C and D) and random cell migration on matrigel was measured using time lapse microscopy (F and G). Tracks of individual migrating cells (n = 60 for each condition) used to measure random cell migration are shown in E. Migration rates (A–D and F–G) are shown as means ± SD. *p < 0.05, **p < 0.01 (Student's t-test, relative to untreated cells).

Fig. 5. Treatment with MnP and dox reduces MDA-MB-231 cell invasion and extracellular proteolytic activity. MDA-MB-231 cells were seeded on matrigel-coated transwells and were allowed to invade for 16 h in the presence of the indicated drugs (dox (0.1 μM), MnP (5 μM)) (A). The percentage of invading cells is summarized in B. The extracellular proteolytic activity was measured using a fluorescent gelatine degradation assay (C). Invasion rates (B), and normalized gelatine degradation (D) from at least three independent experiments are shown as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 (Student's t-test, relative to untreated cells).

Fig. 6. Co-treatment with MnP and dox increases cell area. MCF7 and MDA-MB-231 cell spread on matrigel was monitored over time (A and D). Cells treated with the indicated drugs for 16 h (B and E) or 30 min (C and F) were seeded on matrigel-coated transwells and allowed to spread. The cells were left to adhere for 12 h and cell area was measured. Data is summarized in B, C, E and F. Cell area determined from at least three independent experiments (n > 50 cells per condition and per experiment) and are shown as means ± SEM. *p < 0.05, **p < 0.01 (one-way ANOVA with Tukey's test, relative to untreated cells).

Fig. 7. Effect of MnP and dox on the number of focal adhesions. Confocal images show MCF7 (A) and MDA-MB-231 (C) cells treated with the indicated drugs for 16 h, fixed and stained with anti pFAK. Images are typical of three independent experiments. Scale bars, 20 µm. Summary results (means ± SEM from ≥90 cells for each condition) show numbers of focal adhesions per cell, determined by counting pFAK positive spots (B and D). *p < 0.05, **p < 0.01 , ***p < 0.001 (one-way ANOVA with Tukey's test, relative to untreated cells).

Fig. 8. Effect of MnP and dox treatment on the levels of FA proteins. Typical IB showing total pFAK, FAK, Paxillin and Vinculin and the loading controls (GAPDH and Tubulin) for MCF7 and MDA-MB-231 cells treated with the indicated drugs for 16 h (A). Summary results (means ± SD from three independent experiments) show relative protein expression levels for MCF7 cells (B) and MDA-MB-231 cells (C).

Fig. 9. Effect of MnP and dox treatment on NF-κB-dependent transcription. MCF7 (A) and MDA-MB-231 (B) cells transfected with a firefly luciferase reporter plasmid under the control of an NF-κB-dependent promoter and a renilla luciferase transfection control were treated with the indicated drugs for 16 h. Data are from one experiment representative of at least three, each performed in 5 replicates are presented as means ± SD, *p < 0.05; **p < 0.01; ***p < 0.001 (one-way ANOVA with Tukey's test), compared with untreated cells (-ve).