For this purpose, control and PXR transfected LS174T clones were maintained for 72 hours in the presence of increasing concentrations of irinotecan, SN38, 5 fluorouracil or oxaliplatin. As shown in figure 3, PXR expression clearly led to an increased survival of LS174T cells towards irinotecan and SN38, and the level of chemoresistance to SN38 was correlated inhibitor Alisertib to the relative PXR expression level of each clone. Upon treatment with 1 uM SN38, PXR transfected cells displayed a 6 to 9 fold higher viability compared to pcDNA3 transfected cells. We found no difference in topoisome rase I activity between pcDNA3 Inhibitors,Modulators,Libraries transfected cells and PXR transfected cells demonstrating that the observed chemoresistance is not due to a variation of topoi somerase I expression after PXR transfection As expected, cells were more sensitive to SN38 than to irinotecan, the latter undergoing Inhibitors,Modulators,Libraries minimal conversion into its active metabolite due to very low expression level of carboxylesterases in these cells.

In addition, cell viability Inhibitors,Modulators,Libraries assays performed in SW480 and SW620 cell lines stably transfected with hPXR, showed similar PXR dependent enhancement of SN38 resistance. On the other hand, we found no effect of PXR expression on cell sensitivity to both 5 fluorouracil and oxaliplatin sensitivities. Surprisingly, we observed that rifampicin did not enhance the resistance of cells to SN38. These observations suggest that activation of PXR is not required for these effects or that PXR is already acti vated under these conditions.

While Inhibitors,Modulators,Libraries neither SN38 nor irinotecan activate PXR, we found that PXR was activated in our assays because of the pre sence of 10% fetal calf serum, as previously observed in the HepG2 cell line. Accordingly, in presence of serum, CYP3A4 is highly expressed in PXR2 cells, with no additional effect Inhibitors,Modulators,Libraries of rifampicin, in contrast to excellent validation what we observed in absence of serum. Moreover, we found that the pharmacological PXR inhibitor L sul foraphane decreased the percentage of cell survival in PXR2 cells treated with 1 or 5 uM SN38, compared to cells treated with SN38 alone. Although we cannot completely exclude off target effects of SFN, it is likely that inhibi tion of PXR by SFN contributes toward decreased PXR2 cell resistance. Inhibition of PXR expression reverses chemoresistance to SN38 Because SFN is known to affect several other signaling pathways, such as those involving the transcription fac tors Nrf2 and NF kappaB that are known to affect cell sensitivity to cytotoxics, we then used a more specific strategy to inhibit PXR expression. For this pur pose, LS174T CTRL or PXR2 cells were transduced with control or shRNA expressing lentiviral vectors as described in the material and methods section.