Rapamycin Plus Doxycycline Combination Affects Growth Arrest and Selective Autophagy-Dependent Cell Death in Breast Cancer Cells

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.

Characterization of Mitochondrial Proteome and Function in Luminal A and Basal-like Breast Cancer Subtypes Reveals Alteration in Mitochondrial Dynamics and Bioenergetics Relevant to Their Diagnosis

“…This preference for glycolytic metabolism in both cell lines of BC may be due to a decrease in the mitochondrial respiratory capacity, which was more evident in basal-like cell line by a marked decrease in mitochondrial respiration and decoupling of oxidative phosphorylation compared to luminal A cell line (Figure 6B,C). Concerning these mitochondrial alterations, here, through proteomic analysis, we showed a unique OXPHOS signature capturing the differential expression of complexes proteins between luminal A and basal-like tumors (Figure 10C), which could be of great use in exploring new therapeutic targets for BC [69]. This signature demonstrates a lower expression mainly of complexes I and V in basallike tumors than luminal A tumors.…”

Characterisation of 3D Bioprinted Human Breast Cancer Model for In Vitro Drug and Metabolic Targeting

“…In our previous study, we compared the metabolic enzyme expression of 2D BC cell lines grown in vitro and mTOR, GLS, glycolysis, and lipid metabolism inhibitor sensitivity. These results highlight that metabolic inhibitor combinations-e.g., rapamycin and doxycycline treatments-could have significant antiproliferative effects in in vitro 2D cultured BC cell lines [40]. Therapy resistance could occur and, as a consequence, the drug sensitivity of cancer cell lines could be decreased with a higher passage number in in vitro cultured cells or in xenografts in vivo [41].…”

“…where Ea and Eb represent the detected effect of single monotreatments and Eab indicates the effect of the combined treatment. The assessed CI values can be used for classifying the interactions of treatment agents as follows: (a) synergistic effect (CI < 1); (b) additive effect (CI = 1); and (c) no additional effect (CI > 1) [40,97]. The formation of 3D spheroids was induced by two methods: hanging drop (HD) technique and ultra-low attachment (ULA) plates.…”

Green Tea Epigallocatechin-3-Gallate Regulates Autophagy in Male and Female Reproductive Cancer

“…It responds to a range of cellular stresses, including nutrient deprivation, organelle damage, and abnormal protein accumulation. Autophagy inducers may be beneficial when there are excess abnormal macromolecules and organelles ( Mizushima and Levine, 2020 ), for example, Rapamycin in breast cancer ( Danko et al, 2021 ); Trastuzumab in HER2 positive breast cancer ( Maycotte and Thorburn, 2014 ); Atorvastatin in prostate cancer ( Farrow et al, 2014 ); Metformin in cervical, breast and endometrial cancer ( Lu G. et al, 2021 ), etc .…”