MTA exerts its activity in different pathways increasing the apoptotic stimulus.

<p>Firstly, there is an accumulation of the AMP analogue ZMP that induces the activation of the AMPK pathway, starting a cascade of signalling that affects mTOR and PI3P/Akt pathways; mTOR is inactivated and the accumulation of its downstream unphosphorylated substrates facilitates the apoptosis process. Akt also remains inactive, unable to block p53 and to activate mTOR. On the other hand, the inhibition of TS, DHFR, GARFT and AICART induces oxidative stress and DNA damage which in turn is detected by p53 and caspase-dependent and independent mitochondrial apoptosis that is activated as has been previously reported. Together all processes lead to an imbalance between cell death and survival stimuli that result in enhanced apoptotic signalling.</p

Viability and proliferation XTT after 48 h of exposure to MTA alone or in combination with dTh, Hx, or/and AICA.

<p>The percentage of viable cells is shown relative to viability of MTA-unexposed cells (control conditions). These results are representative of three independent experiments. <b>A)</b> Viability assays before and after MTA exposure with the pyrimidine biosynthesis pathway restored by addition of Hx alone or in combination with dTh. <b>B)</b> Viability assays before and after MTA exposure with purine biosynthesis pathway restored through the addition of AICA alone or in combination with dTh. <b>C)</b> Heatmap of six MTA-related genes where up- and down-regulation fold changes corresponding to each colour are indicated on the scale on the right of the figure.</p

A schematic diagram of the pyrimidine and purine biosynthesis pathways is shown, where the MTA-targets are indicated with a lightning bolt symbol.

<p>The effect of the addition to the culture medium of the preformed purine Hx, the preformed pyrimidine dTh and/or the purine pathway intermediate AICA as a new source for the restoration of the MTA-interrupted biosynthesis of purines and pyrimidines was different depending on the cell line.</p

Table summarizing the genetic characteristics of studied cell lines.

<p>According to COSMIC database from <b>Sanger Institute</b> and “<b>The TP53 web site</b>”.</p

Medians of overall survival (months) for the three genes, <i>ERS1</i>, <i>ERS2</i>, and <i>CYP19A1</i> (Low and High, N = 48) and aromatase protein, ARO (Low N = 43 and High N = 42), when categorizing their values into low and high expression values (lower and higher expression than median).

<p>Kaplan-Meier Log Rank and Mann-Whitney p-values when comparing Low vs. High groups are also listed. T = Tumor; NT = Non-tumor.</p><p>Medians of overall survival (months) for the three genes, <i>ERS1</i>, <i>ERS2</i>, and <i>CYP19A1</i> (Low and High, N = 48) and aromatase protein, ARO (Low N = 43 and High N = 42), when categorizing their values into low and high expression values (lower and higher expression than median).</p

Kaplan-Meier survival curves when considering gene mRNA expression levels (<i>ERS1, ERS2</i> and <i>CYP19A1</i>), or protein concentration (ARO), as discriminating factor.

<p>The patient sample is divided into Low and High (lower and higher expression than median). Panels: a) <i>ERS1</i>and <i>ERS2</i>, b) <i>CYP19A1</i> and ARO, and c) <i>ERS1+ERS2</i> and CYP19A1 + ARO.</p

Medians for the three genes (<i>ERS1</i>, <i>ERS2</i>, and <i>CYP19A1</i>) relative expression (RQ) measures, N = 96, and aromatase protein concentration, N = 85.

<p>Also includes p-values for Man-Whitney tumor/non-tumor RQ comparison.</p><p>T = Tumor; NT = Non-tumor.</p><p>ARO* =  Protein concentration ELISA values, ng/ml.</p><p>Medians for the three genes (<i>ERS1</i>, <i>ERS2</i>, and <i>CYP19A1</i>) relative expression (RQ) measures, N = 96, and aromatase protein concentration, N = 85.</p

Statistics of overall survival (months) of tumor samples for <i>ERS1</i> gene expression, <i>ERS2</i> gene expression, <i>CYP19A1</i> gene expression and aromatase protein concentration (ARO) for each combination of low and high expression groups (lower and higher than median).

<p>Kaplan-Meier Log Rank (Mantel-Cox) and Kruskal-Wallis p-values are listed when comparing these different categories. N: Patient number.</p><p>Statistics of overall survival (months) of tumor samples for <i>ERS1</i> gene expression, <i>ERS2</i> gene expression, <i>CYP19A1</i> gene expression and aromatase protein concentration (ARO) for each combination of low and high expression groups (lower and higher than median).</p

Patient Characteristics.

<p>*<i>Chemotherapy.</i></p><p>Patient Characteristics.</p