Simultaneous Analysis of Wnt and NF-κB Signaling Pathways in Doxorubicin Sensitive and Methotrexate Resistant PLC/ PRF/5 Cells.

Abstract

Multi-drug resistance (MDR) is a controversial issue in traditional chemo- therapy of aggressive cancers, including hepatocellular carcinoma. The major cause of MDR is suggested to be the aberrant activation of the main signaling pathways such as Wnt and nuclear factor kappa-light-chain-enhancer of activated B cells (NF- κB) which have key roles in the maintenance of cancer stem cells (CSCs). Therefore, the evaluation of their alterations could be essential in chemo-resistant cancers such as Hepatocellular carcinoma. The main purpose of this study was to investigate the alteration of the mentioned pathways in the chemotherapy resistant cancer cells by assessing their major molecular parameters. In this experimental study, methylthiazol tetrazolium (MTT) assay, acridine orange/ethidium bromide (AO/EtBr) and Hoechst 33342 staining, DNA fragmentation and colony formation methods were employed to investigate the cytotoxic effects of methotrexate (MTX) and doxorubicin (DOX) on PLC/PRF/5 cells. Moreover, the expression of 11 important genes involved in MDR was performed by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). PLC/PRF/5 cells (Alexander) were sensitive to DOX and normally resist- ant to MTX. In addition, the results obtained from RT-PCR analysis revealed that β-catenin expression was significantly reduced and ABCG2 significantly overex- pressed 4.85 and 3.34 times (P value<0.05) in DOX and MTX treated cells, respec- tively. Furthermore, a considerable expression of HIF-1α and p65 were detected only in MTX-resistant cells. Anti-cancer drugs may have more than one target in tumor cells. They not only participate in deregulation of Wnt but also alter NF-κB activation. Moreover, HIF-1α was the only anti-apoptotic protein that was significantly induced in the chem- oresistant cells.