Mitochondrial division inhibitor (mdivi-1) induces extracellular matrix (ECM)-detachment of viable breast cancer cells by a DRP1-independent mechanism

Abstract

Increasing evidence supports the hypothesis that cancer progression is under mitochondrial control. Mitochondrial fission plays a pivotal role in the maintenance of cancer cell homeostasis. The inhibition of DRP1, the main regulator of mitochondrial fission, with the mitochondrial division inhibitor (mdivi-1) had been associated with cancer cell sensitivity to chemotherapeutics and decrease proliferation. Here, using breast cancer cells we find that mdivi-1 induces the detachment of the cells, leading to a bulk of floating cells that conserved their viability. Despite a decrease in their proliferative and clonogenic capabilities, these floating cells maintain the capacity to re-adhere upon re-seeding and retain their migratory and invasive potential. Interestingly, the cell detachment induced by mdivi-1 is independent of DRP1 but relies on inhibition of mitochondrial complex I. Furthermore, mdivi-1 induces cell detachment rely on glucose and the pentose phosphate pathway. Our data evidence a novel DRP1-independent effect of mdivi-1 in the attachment of cancer cells. The generation of floating viable cells restricts the use of mdivi-1 as a therapeutic agent and demonstrates that mdivi-1 effect on cancer cells are more complex than anticipated.

Keywords: Cancer; Cell detachment; Mdivi-1; Metabolism; Mitochondrial complex I.

Figure 1

Mdivi-1 induces detachment of viable breast cancer cells. (a) MCF-7 and MDA-MB-231 cells were photographed, and the cellular morphology was documented using a phase-contrast microscope. (b) Schematic representation of the separation of floating and adherent cells into four cell populations: FC, FM, AC, and AM (Created with BioRender.com). (c) MCF-7 and (d) MDA-MB-231 cells were treated with 50 μM mdivi-1 for 24 h. Then, the fraction of viable floating cells relative to the total viable cell population and viable adherent cells was determined. (e), (f) MCF-7 and (e), (g) MDA-MB-231 cells were treated with 50 μM mdivi-1 for 24 h. Next, a clonogenic assay was performed using only the floating cells, FC and FM, isolated from the cell culture medium. FC and FM cells were re-seeding and resuspended in mdivi-1-free cell culture medium. After seven days of growth, the number of colonies were measured using ImageJ software. The scale bar corresponds to 100-μm. Results represent means ± SEM. *p < 0.05; **p < 0.01; ****p < 0.0001.

Figure 2

The floating breast cancer cells induced by mdivi-1 maintain the proliferative and clonogenic potential upon re-seeding. (a), (b) MCF-7 and (c), (d) MDA-MB-231 cells were treated with 50 μM mdivi-1 for 24 h. Floating and adherent cancer cells were collected and re-seeded in mdivi-1-free cell culture medium for 24, 48, 72, and 96 h. Then, a proliferation assay was performed using crystal violet. (e), (f) MCF-7 and (g), (h) MDA-MB-231 cells were treated with 50 μM mdivi-1 for 24 h. Next, four cell populations were collected and re-seeding to carry out the clonogenic assay. After seven days of growth, the number and area of colonies were measured using ImageJ software. (i) MCF-7 and (j) MDA-MB-231 cells were treated with 50 μM mdivi-1 for 24 h. Floating and adherent cells were collected, and then a cell cycle assay analysis was performed. Results represent means ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 3

The floating breast cancer cells induced by mdivi-1 maintain the migratory potential upon re-seeding. (a) MDA-MB-231 and (b) MCF-7 cells were treated with 50 μM mdivi-1 for 24 h. Floating and adherent cells were collected, and a transwell migration assay was performed. The four cell populations were photographed, and in vitro transmigration was documented using a phase-contrast microscope. Results represent means ± SEM. **p < 0.01; ns = not significant.

Figure 4

Mdivi-1 induces detachment of breast cancer cells by a DRP1-independent mechanism. (a) DRP1 and β-actin protein levels in WT and DRP1-KO HCT116 cells were determined by western blot (b) these cells were treated with 50 μM mdivi-1 for 24 h. Next, the fraction of viable floating cells relative to the total viable cell population was determined. (c) MCF-7 and MDA-MB-231 cells were treated with 50 μM mdivi-1 for 4 h. Then, a Mito Stress test was performed using a seahorse XFe96 analyzer. (d) MCF-7 and (e) MDA-MB-231 cells were treated with 10 μM rotenone for 24 h, and then, the fraction of viable floating and adherent cells was determined. (f) MCF-7 and MDA-MB-231 cells were treated with 5 μM antimycin-A (AA) and 5 μM oligomycin (Oligo) for 24 h. The fraction of viable floating cells was determined. (g) 143Bwt, 143BΔcytb and (h) Rho-0 cells were treated with 50 μM mdivi-1 for 24 h. Then, the fraction of viable floating and adherent cells was determined. Results represent means ± SEM. *p < 0.05; **p < 0.01; ns = not significant.

Figure 5

Mdivi-1 induces cell detachment in a glucose-dependent manner. (a) MDA-MB-231 cells were treated with 50 μM mdivi-1 and 10 μM rotenone for 24 h. Floating and adherent cells were collected, and a glucose uptake was assessed using the fluorescent substrate 2-NBDG. (b) MCF-7 and MDA-MB-231 cells were treated with 50 μM mdivi-1 for 24 h in a glucose-free cell culture medium. The fraction of viable floating cells was determined. (c) The oxidative subpopulation of MDA-MB-231 cells were treated with 50 μM mdivi-1 for 24 h. Next, the fraction of viable floating and adherent cells was determined. (d) MCF-7 and MDA-MB-231 cells were treated with 50 μM mdivi-1 plus 5 μM BAY-876 for 24 h. Then, the fraction of viable floating cells was determined. (e) MCF-7 and MDA-MB-231 cells were treated with mdivi-1 for 24 h in a glucose-free cell culture medium with 25 mM fructose. The fraction of viable floating cells was determined. (f) MCF-7 and MDA-MB-231 cells were treated with 50 μM mdivi-1 plus 50 μM 6-ANA for 24 h. The fraction of viable floating cells was determined. (g) MCF-7 and MDA-MB-231 cells were treated with 10 μM rotenone plus 50 μM 6-ANA for 24 h. The fraction of viable floating cells was determined. (h) MCF-7 and MDA-MB-231 cells were treated with 50 μM mdivi-1 plus 50 μM 6-ANA for 24 h. Then, a clonogenic assay was performed using only floating cells isolated from the cell culture medium. Next, cells were re-seeding and resuspended in mdivi-1-free cell culture medium. After seven days of growth, the number of colonies were measured using ImageJ software. Results represent means ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ns = not significant.

Figure 6

Model of detachment of viable human breast cancer cells induced by mdivi-1. The inhibition of mitochondrial complex I by mdivi-1 triggers the detachment of viable breast cancer cells. These cells maintain their proliferative, migratory, and invasive potential once re-seeding. The effect of mdivi-1 on the cell detachment is independent of DRP1 but dependent on the pentose phosphate pathway (PPP) upon complex I inhibition OM: Outer Mitochondrial Membrane, IS: Intermembrane Space, IM: Inner Mitochondrial Membrane, MM: Mitochondrial Matrix, I: Mitochondrial complex I. (Created with BioRender.com).