Abstract 483P
Background
Colorectal cancer (CRC) is one of the most frequently diagnosed malignancies worldwide. Although chemotherapy represents an effective anti-tumour therapeutic approach for CRC, drug treatment efficacy is often limited due to the development of resistant cancer cells. Several studies associate the pregnane X receptor (PXR) and autophagy with resistance to chemotherapeutic drugs. This study aims to investigate the relationship between mtKRAS- dependent autophagy and PXR expression after treatment with irinotecan in CRC.
Methods
MtKRAS cell lines, HCT116, DLD-1 and sw480, were exposed to 10 μΜ Irinotecan and 80nM siRNA-PXR for 48hrs. In addition, the same cell lines were exposed to 5mM 3-MA (3-Methyladenine), 10μΜ HCQ (Hydroxychloroquine) and 1μΜ of PI-103 for 24 hrs. The protein levels of p62, LC3, PARP, and PXR were detected by Western blotting. The viability of cells was measured through MTT analysis. The sub-cellular localization of PXR was detected with confocal microscopy. RT-PCR was used to test the expression levels of different genes after the siRNA silencing of PXR in mtKRAS CRC cell lines.
Results
Increased PXR expression was noted in mtKRAS cell lines. Irinotecan activates autophagy in all CRC cell lines, identified through the increasing ratio of LC3II/I and reduction of p62. After knockdown of PXR with siRNA, autophagy activation remained unchanged and apoptosis was observed. Next, mtKRAS CRC cell lines were treated with two autophagy inhibitors (3-MA and HCQ) and with the dual inhibitor of AKT/mTOR (PI-103). Induction (PI-103) or inhibition (HCQ) of autophagy altered the PXR protein levels (reduced and increased, respectively). Moreover, PXR nuclear distribution was increased post-autophagy induction, as shown by confocal microscopy. Alterations of the expression levels of several genes, such as MDR1, CYP3A4, ABCB1, NR1I3, FGF19, and GADD45B, were observed by RT-PCR in mtKRAS CRC cell lines after the silencing of PXR.
Conclusions
Collectively, these results support the hypothesis that autophagy acts as a cytoprotective mechanism against irinotecan, possibly through the induction and subcellular trafficking of PXR in mtKRAS as observed in CRC models.
Clinical trial identification
Editorial acknowledgement
Legal entity responsible for the study
Theocharis Stamatios.
Funding
Has not received any funding.
Disclosure
All authors have declared no conflicts of interest.