Introduction: Recent years brought many breakthrough discoveries in cancer research and therapy, malignant diseases such as breast cancer still remain one of the major health threats worldwide. Cancer cells usually gain resistance to regulated cell death (RCD) and thus there is a constant need for novel anti-cancer drugs and treatment protocols targeting RCD pathways in cancer cells.

Aims: Among the novel RCD-inducing agents belongs also mitochondria-targeted tamoxifen – MitoTam, which is the major focus of this study with the following aims:

1. Analysis of the expression of RCD-related genes in human breast cancer cells and determination of respiration/glycolysis status.

2. Induction and time-lapse profiling of RCD triggered by MitoTam in breast cancer cells and its modulated by metabolic/energy pathways (MEP) inhibitors and BH3 mimetics.

Methods: In this study we employed the XFe96 Seahorse/Agilent analyser (mitochondrial respiration and cellular glycolysis), Oxygraph2k/Oroboros (mitochondrial respiration), western blotting and time-lapse monitoring of cell proliferation and cell death using Incucyte SX1 and Lumascope LS720 instruments.

Results: With few exceptions, all cell types expressed essential RCD-related proteins and responded to MitoTam treatment with various efficacy. The glycolysis-preferring breast cancer cell lines such as MDA-MB-231 are more resistant to MitoTam treatment than mitochondrial respiration-based MDA-MB-453 cells. The co-treatment experiments showed strong enhancing effect of Bcl-XL inhibitor A1155463 on MitoTam-induced RCD in MDA-MB-231 and T47D cells and of Mcl-1 inhibitor S63845 on MDA-MB-453 cells. Selected MEP inhibitors such as the lactate dehydrogenase inhibitor (R)-GNE-140 or pyruvate dehydrogenase kinases inhibitor JX06 greatly enhanced MitoTam-induced RCD of breast cancer cells.

Conclusions:

1. Breast cancer cells with higher levels of mitochondrial respiration appeared to be more sensitive to MitoTam treatment.

2. Breast cancer cells resistant to MitoTam-induced RCD can be sensitized by their co-treatment with (a) BH3 mimetics targeting Bcl-XL or Mcl-1 or (b) selected MEP inhibitors preferentially targeting glycolysis-affecting/modulating signalling.