Mechano-induced cell metabolism disrupts the oxidative stress homeostasis of SAOS-2 osteosarcoma cells

Abstract

There has been an increasing focus on cancer mechanobiology, determining the underlying-induced changes to unlock new avenues in the modulation of cell malignancy. Our study used LC-MS untargeted metabolomic approaches and real-time polymerase chain reaction (PCR) to characterize the molecular changes induced by a specific moderate uniaxial stretch regimen (i.e., 24 h-1 Hz, cyclic stretch 0,5% elongation) on SAOS-2 osteosarcoma cells. Differential metabolic pathway analysis revealed that the mechanical stimulation induces a downregulation of both glycolysis and the tricarboxylic acid (TCA) cycle. At the same time, the amino acid metabolism was found to be dysregulated, with the mechanical stimulation enhancing glutaminolysis and reducing the methionine cycle. Our findings showed that cell metabolism and oxidative defense are tightly intertwined in mechanically stimulated cells. On the one hand, the mechano-induced disruption of the energy cell metabolism was found correlated with an antioxidant glutathione (GSH) depletion and an accumulation of reactive oxygen species (ROS). On the other hand, we showed that a moderate stretch regimen could disrupt the cytoprotective gene transcription by altering the expression levels of manganese superoxide dismutase (SOD1), Sirtuin 1 (SIRT1), and NF-E2-related factor 2 (Nrf2) genes. Interestingly, the cyclic applied strain could induce a cytotoxic sensitization (to the doxorubicin-induced cell death), suggesting that mechanical signals are integral regulators of cell cytoprotection. Hence, focusing on the mechanosensitive system as a therapeutic approach could potentially result in more effective treatments for osteosarcoma in the future.

Keywords: cyclic stretch; mechanobiology; metabolomics (LC-MS); osteosarcoma; oxidative stress response.

FIGURE 1

Volcano plot showing the distribution of the fold changes in metabolite concentrations induced by the 24 h 1 Hz cyclic stretch of SAOS-2 cells. Blue and red dots refer to decreased and increased metabolites, respectively, in the 1 Hz samples (fold change >1.5 and adjusted p-value FDR < 0.05).

FIGURE 2

Metabolomics Pathway Analysis (MetPA) of mechanically 1 Hz stimulated SAOS-2 cell line. The color of each circle is based on p-values (darker colors indicate more significant changes of metabolites in the corresponding pathway). In contrast, the circle size corresponds to the pathway impact score. The most impacted pathways (high statistical significance scores) are annotated by their full name.

FIGURE 3

Changes in accumulation of intermediates belonging to glycolysis and Pentose Phosphate Pathway in mechanically stimulated (1 Hz) samples compared to the control (Ctrl). Data are presented as mean ± standard deviation. The statistical significance of each metabolite between samples was determined using Student’s t-test. *p-value < 0.05; **p-value < 0.01; ***p-values < 0.001.

FIGURE 4

Changes in accumulation of TCA intermediates and glutaminolysis in mechanically stimulated (1 Hz) samples compared to the control (Ctrl). Data are presented as mean ± standard deviation. The statistical significance of each metabolite between samples was determined using Student’s t-test. *p-value < 0.05; **p-value < 0.01; ***p-values < 0.001.

FIGURE 5

Mechanically-induced changes in the accumulation of intermediates belonging to aminoacid and vitamin B6 metabolisms. Metabolite quantification is reported for the mechanically stimulated SAOS-2 cells (1 Hz) and the static control counterpart (Ctrl). Data are presented as mean ± standard deviation. The statistical significance of each metabolite between samples was determined using Student’s t-test. *p-value < 0.05; **p-value < 0.01; ***p-values < 0.001. The bold arrow indicates the reaction that requires vitamin B6 as a cofactor.

FIGURE 6

Mechanically-induced changes in the accumulation of intermediates belonging to pyrimidine and purine metabolisms in mechanically stimulated (1 Hz) samples compared to the control (Ctrl). Data are presented as mean ± standard deviation. The statistical significance of each metabolite between samples was determined using Student’s t-test. *p-value < 0.05; **p-value < 0.01; ***p-values < 0.001.

FIGURE 7

Mechanically-induced upregulation of ROS and Cytotoxicity in SAOS-2 cell line. (A) Cellular ROS production for the 1 Hz-stimulated or not stimulated cells is reported both in the presence and the absence of 8,6 µM Doxorubicin treatment. (B) Doxorubicin Cytotoxicity for SAOS-2 osteosarcoma cells that were or were not pre-mechanically treated. Light green square symbols represent relative cell viability for cells cyclically stimulated for 24 h at 1 Hz frequency. Dark-green triangles represent control static cells (cells cultured on a silicone support subjected to the same experimental conditions but not stretched). Statistics have been performed on three biological replicates with four technical replicates per condition. (C) The impact of a 24 h 1 Hz uniaxial stimulation on the gene expression of four cytoprotective genes (i.e., SOD1, SIRT1, Nrf2, and FOXO-1). Statistical analyses were performed on three biological replicates with at least three technical replicates per condition. Statistical significance between treated and control samples was determined using Student’s t-test. *p-value < 0.05; **p-value < 0.01.

FIGURE 8

Schematic Illustration of the stretch-induced changes in SAOS-2 cells: Upper panel. Cell Morphology: Our previous findings using confocal and atomic force microscopy (AFM) revealed that the application of cyclic stretch induces significant alterations in the morphology of SAOS-2 cells (Alloisio et al., 2023). Notably, the nucleus area, recognized for its crucial role in mechano-regulating cell behaviors, undergoes enlargement. The entire cell undergoes increased elongation post-cyclic stretch application, resulting in a noteworthy increase in size and disruption of the nuclear-to-cellular (N/C) ratio of SAOS-2 cells (Alloisio et al., 2023). Lower panel. Gene Expression Levels: While the expression levels of osteogenic differentiative markers (i.e., ALP, COL1, RUNX-2) remain unaffected by mechanical stimulation (Alloisio et al., 2023), the present data shows that SIRT1 and Nrf2 are downregulated, whereas SOD1 is upregulated in the mechanically treated samples compared to the untreated specimens. Metabolic State: Mechanical stimulation is associated with a reversal of the Warburg effect, upregulation of glutaminolysis, GSH depletion, intracellular ROS accumulation, and upregulation of PPP, coupled with downregulation of nucleotide synthesis.