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.
Highlights
We found that the rapamycin + doxycycline combination could decrease the cell proliferation capacity in either an additive or synergistic manner in about 2/3rds of the investigated cell lines (Figure 1a)
We show that rapamycin combined with doxycycline has considerable tumour growth inhibitory effects in different cells with various origins
Our results highlight that long-term/continuous doxycycline combined with rapamycin significantly decreased or even stopped cell proliferation leading to a degrading different cell culture system, especially in breast cancers
Summary
Considering the diagnosed malignancies, breast cancer is one of the most frequent neoplasms, with an elevating incidence rate [1]. Developing resistance during common and targeted therapies is in correlation with the failures of cancer care. Mammalian target of rapamycin (mTOR) complexes are in a central regulatory position in signalling networks, including metabolic rewiring in the course of tumour development. MTOR hyperactivity is characteristic for many cancers, especially in the worst cases [2,3]. Rapamycin analogues (rapalogs) were introduced into the therapy of many cancers (e.g., advanced breast cancers), but the already tested rapalogs could not achieve breakthrough success in cancer treatments [4]. As regards, targeting metabolic processes after or simultaneously with different drug administrations could be an additional option in future therapy developments [7,8]
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