Abstract
The metabolic shift induced by hypoxia in cancer cells has notbeen explored at volatilomic level so far. The volatile organic metabolites (VOMs) constitute an important part of the metabolome and their investigation could provide us crucial aspects of hypoxia driven metabolic reconfiguration in cancer cells. To identify the altered volatilomic response induced by hypoxia in metastatic/aggressive breast cancer (BC) cells. BC cells were cultured under normoxic and hypoxic conditions and VOMs were extracted using HS-SPME approach and profiled by standard GC-MS system. Univariate and multivariate statistical approaches (p < 0.05, Log2 FC ≥ 0.58/≤ - 0.58, PC1 > 0.13/< - 0.13) were applied to select the VOMs differentially altered after hypoxic treatment. Metabolic pathway analysis was also carried out in orderto identify altered metabolic pathways induced by the hypoxia in the selected BC cells. Overall, 20 VOMs were found to besignificantly altered (p < 0.05, PC1 > 0.13/< - 0.13) upon hypoxic exposure to BC cells. Further, cell line specific volatilomic alterations were extracted by comparative metabolic analysis of aggressive (MDA-MB-231) vs. non-aggressive (MCF-7) cells incubated under hypoxia and normoxia. In this case, 15 and 12 VOMs each were found to be significantly altered in aggressive cells when exposed to hypoxic and normoxic condition respectively. Out of these, 9 VOMs were found to be uniquely associated with hypoxia, 6 were specific to normoxia and 6 were found common to both the conditions. Formic acid was identified as the most prominent molecule with higher abundance levels in aggressive as compared to non-aggressive cells in both conditions. Furthermore, metabolic pathway analyses revealed that fatty acid biosynthesis and nicotinate and nicotinamide metabolism were significantly altered in aggressive as compared to non-aggressive cells in normoxia and hypoxia respectively. Higher formate overflow was observed in aggressive cells compared to non-aggressive cells incubated under both the conditions, reinforcing its correlation with aggressive and invasive cancer type. Moreover, under hypoxia, aggressive cells preferred to be bioenergetically more efficient whereas, under normoxia, fatty acid biosynthesis was favoured when compared to non-aggressive cells.
Highlights
The metabolic shift induced by hypoxia in cancer cells has not been explored at volatilomic level so far
Multivariate statistical analysis such as Principal Component Analysis (PCA) was carried out in order to visualise the pattern associated with the hypoxia-induced metabolic changes (Fig. 1).The PCA score plots depict the separation of the four groups belonging to non-aggressive and aggressive cells grown under normoxia and hypoxia (Fig. 1a and b).The segregation of the groups in PCA model indicate that metabolic differences are dependent of the cell line and growing conditions
Hypoxia induces distinct volatilomic responses in breast cancer (BC) cells which can be efficiently extracted, analyzed and identified by Headspace Solid-Phase Microextraction (HS-SPME) coupled to GC–MS
Summary
The metabolic shift induced by hypoxia in cancer cells has not been explored at volatilomic level so far. Cell line specific volatilomic alterations were extracted by comparative metabolic analysis of aggressive (MDA-MB-231) vs non-aggressive (MCF-7) cells incubated under hypoxia and normoxia. In this case, 15 and 12 VOMs each were found to be significantly altered in aggressive cells when exposed to hypoxic and normoxic condition respectively. Hypoxic condition induces several adaptive responses in the cancer cells which allow them to adjust to hostile environment (Vaupel 2004). These effects are carried out by the group of transcription factors known as hypoxia inducible factors (HIF), which comprise of the α and β heterodimers viz. HIF-1α, HIF-2α and HIF-3α (Prabhakar and Semenza 2012; Semenza 2012). HIF1α is considered as a master regulator of the hypoxia-driven responses that are critical for the malignant features of the tumor (Semenza 2010a, b)
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