Franny Jongbloed

56 CHAPTER 3 irinotecan comparison. Of these 754 genes, 459 DEPS were downregulated and 295 were upregulated. To analyze common expression patterns between the AL and fasting groups, the overlapping DEPS were visualized using a Venn diagram and a scatterplot. The Venn diagramshowed that 329DEPS overlapped between the AL irinotecan and fasting irinotecan groups, corresponding to 44% of the DEPS found after fasting, and only 14% of the DEPS after AL vehicle vs. AL irinotecan (Figure 2C). Directionality and expression intensity of the unique and overlapping genes were visualized in a scatterplot. This scatterplot showed that 83% of all DEPS had a similar directionality in both comparisons, but fold change expressions of the AL irinotecan groups (red symbols) were on average higher than the fasting irinotecan groups (green symbols). Of the overlapping DEPS (black symbols), 99% had a similar directionality (Figure 2D). Collectively, these results point towards a dampened response upon irinotecan exposure in the fasted group compared to that found in AL fed mice. Pathway analyses To explore the pathways regulated by irinotecan, the 2,667 DEPS resulting from the comparison of AL vehicle with AL irinotecan were analyzed. A total of 20 pathways were found to be regulated, defined as pathways with a significant P- value of <0.05 and a z-score of ≥1 or ≤-1 (Table 1A). Of these 20 pathways, 17 were activated and three were inhibited. Analysis of these pathways resulted in an image of an activated genotoxic response. This genotoxic response revealed itself via the activation of multiple pathways involved in intracellular and second messenger signaling, including the RhoA DNA damage pathway. Both cellular stress and cytokine signaling pathways were activated as well, of which most notable eIF2 and interleukin signaling. Apoptosis was stimulated via activation of MAPK signaling. A similar pathway analysis between the fasting groups was performed after exposure to irinotecan which revealed 39 pathways, of which 35 were downregulated and four were upregulated (Table 1B). In contrast to the response in the AL fed animals, an overall inhibition of the response was shown. Comparison of the regulated pathways between the AL groups and the fasting groups revealed a near opposite image (Table 1). Especially cytokine signaling and cellular immune response appeared downregulated, including chemokine signaling and the IL-8 signaling pathway. Irinotecan exposure in a fasting state also resulted in an inhibition of cellular growth and proliferation, including the inhibition of P70S6K signaling, indicating a decrease in activity of the nutrient-sensor mammalian target of rapamycine (mTOR). Xenobiotic metabolism appears reduced via an inhibition of the aryl hydrocarbon receptor (AHR) pathway, while the stress resistance inducing NRF2-pathway was upregulated.