Figure 1.
Doxycycline alters the metabolic gene expression profile of MCF12A cells.
Treatment of MCF12A cells with Dox at a concentration of 1 µg/mL shows widespread changes in expression of metabolic genes. A) GSEA reveals the most significantly altered metabolic pathways, ranked by normalized enrichment score (NES), in Dox treatment compared to vehicle. KEGG pathway entries are denoted in parentheses where appropriate. Pathways without KEGG entries–All Metabolic Genes and Glycolysis-Gluconeogenesis & Pentose Phosphate–are artificial combinations of other pathways with redundant genes collapsed. All Metabolic Pathways includes all non-redundant genes from every KEGG pathway analyzed. B) This heat map highlights changes in the constituent genes of the oxidative phosphorylation and glycolysis/gluconeogenesis/pentose phosphate pathways upon treatment. Annotated genes include those encoding regulatory enzymes in glycolysis (phosphofructokinase (PFK), hexokinase (HK), pyruvate kinase (PK), shown in blue) and in gluconeogenesis (glucose-6-phosphatase (G6PC) and fructose-1,6-bisphosphatase (FBP), shown in orange). (C) Altered expression of regulatory enzymes in glycolysis and its proximal carbon shunts are shown schematically, with red indicating upregulation and green indicating downregulation.
Figure 2.
Tetracycline antibiotics affect glucose metabolism and oxygen consumption in a dose-dependent fashion.
Effects of the tetracyclines on glycolytic flux were determined by measuring changes in A) glucose consumption and B) lactate production rates of MCF12A cells when treated with Dox, Mino, or Tet at 100 ng/mL or 1 µg/mL. A dose-response relationship was established between Dox and C) lactate production rate in MCF12A cells, D) lactate production rate in 293T cells, E) oxygen consumption rate in MCF12A cells, and F) oxygen consumption rate in 293T cells by treating with a range of drug concentrations. Error bars represent standard deviation from experimental triplicate measurements for all assays, except for oxygen consumption dose-response in 293T cells, which was performed with 8 replicates. * denotes a p-value ≤0.05, ** a p-value ≤0.01 from a two-tailed Student’s t-test.
Figure 3.
Doxycycline can alter both glycolytic and oxidative metabolism in a heterogeneous panel of human cell lines.
Findings were extended to a panel of human cell lines, which were treated with Dox for 96 hours at 100 ng/mL, 1 µg/mL, or with vehicle control and then assayed for A) glucose consumption rate, B) lactate production rate, and C) basal oxygen consumption rate. D) To assess the temporality of this phenotype, MCF12A cells were treated with Dox at 1 µg/mL at shorter time points–24, 48, and 72 hours–and oxygen consumption rate was measured. Error bars represent standard deviation from experimental triplicate measurements in 3A and 3B; from 6 (100 ng/mL) or 7 (control, 1 µg/mL) replicates in 3C; and from 5 replicates in 3D. Oxygen consumption measurements for 293T cells from Figure 2F were repurposed for 3C. * denotes a p-value ≤0.05, ** a p-value ≤0.01 from a two-tailed Student’s t-test.
Figure 4.
Doxycycline reduces proliferation of multiple human cell lines.
Cell numbers were measured over 96 hours of Dox treatment. Shown here are representative growth curves for A) MCF12A and B) 293T cells, as well as C) the full panel of cell lines with cell counts after 96 hours of treatment normalized to vehicle control. To interrogate the reason for the proliferation defect, PI-based cell cycle analysis was performed on D) MCF12A and E) 293T cells, and Annexin V/PI viability analysis was performed on F) MCF12A and G) 293T cells. Both proliferation and flow cytometry-based assays were performed in triplicate, with error bars representing standard deviation. * denotes a p-value ≤0.05, ** a p-value ≤0.01 from a two-tailed Student’s t-test.