Rapamycin Plus Doxycycline Combination Affects Growth Arrest and Selective Autophagy-Dependent Cell Death in Breast
Titanilla Dankó1, Gábor Petővári1, Dániel Sztankovics1
11st Department of Pathology and Experimental Cancer Research, Semmelweis University, Üllői út 26, H-1085 Budapest, Hungary.
Abstract:
Metabolic alteration is characteristic during tumour growth and therapy; however, targeting metabolic rewiring could overcome therapy resistance. mTOR hyperactivity, autophagy and other metabolic processes, including mitochondrial functions, could be targeted in breast cancer progression. We investigated the growth inhibitory mechanism of rapamycin + doxycycline treatment in human breast cancer model systems. Cell cycle and cell viability, including apoptotic and necrotic cell death, were analysed using flow cytometry, caspase activity measurements and caspase-3 immunostainings. mTOR-, autophagy-, necroptosis-related proteins and treatment-induced morphological alterations were analysed by WesTM, Western blot, immunostainings and transmission electron microscopy. The rapamycin + doxycycline combination decreased tumour proliferation in about 2/3rd of the investigated cell lines. The continuous treatment reduced tumour growth significantly both in vivo and in vitro. The effect after short-term treatment was reversible; however, autophagic vacuoles and degrading mitochondria were detected simultaneously, and the presence of mitophagy was also observed after the long-term rapamycin + doxycycline combination treatment. The rapamycin + doxycycline combination did not cause apoptosis or necrosis/necroptosis, but the alterations in autophagy- and mitochondria-related protein levels (LC3-B-II/I, p62, MitoTracker, TOM20 and certain co-stainings) were correlated to autophagy induction and mitophagy, without mitochondria repopulation. Based on these results, we suggest considering inducing metabolic stress and targeting mTOR hyperactivity and mitochondrial functions in combined anti-cancer treatments.
Insights
Combining rapamycin and doxycycline targets metabolic rewiring in breast cancer, inhibiting tumor growth by inducing autophagy and mitophagy. This approach offers a novel strategy to overcome therapy resistance by targeting cellular metabolism.
Area of Science:
- Oncology
- Molecular Biology
- Cellular Metabolism
Background:
- Metabolic alterations are hallmarks of tumor growth and therapy resistance in breast cancer.
- Hyperactivity of mTOR, dysregulated autophagy, and impaired mitochondrial function contribute to breast cancer progression.
Purpose of the Study:
- To investigate the growth inhibitory mechanisms of combined rapamycin and doxycycline treatment in human breast cancer models.
- To explore the impact of this combination therapy on cell cycle, cell viability, cell death pathways, and key molecular targets.
Main Methods:
- Flow cytometry, caspase activity assays, and immunostainings were used to analyze cell cycle, viability, and apoptosis/necrosis.
- Wes™, Western blot, immunostainings, and transmission electron microscopy assessed mTOR, autophagy, necroptosis proteins, and morphological changes.
- In vitro and in vivo models were utilized to evaluate tumor proliferation and growth inhibition.
Main Results:
- Rapamycin + doxycycline decreased tumor proliferation in approximately two-thirds of tested cell lines and significantly reduced tumor growth in vitro and in vivo.
- Long-term treatment induced autophagy and mitophagy, characterized by autophagic vacuoles and degrading mitochondria, without causing apoptosis or necrosis/necroptosis.
- Alterations in autophagy- and mitochondria-related proteins (LC3-B-II/I, p62, MitoTracker, TOM20) correlated with autophagy induction and mitophagy.
Conclusions:
- The combination of rapamycin and doxycycline effectively inhibits breast cancer growth by inducing metabolic stress, specifically targeting mTOR hyperactivity and mitochondrial functions.
- This therapeutic strategy promotes autophagy and mitophagy, offering a potential approach to overcome resistance in breast cancer treatment.
- Inducing metabolic stress and targeting mitochondrial dynamics represent a promising avenue for combined anti-cancer therapies.
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