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Simultaneous Analysis of Wnt and NF-κB Signaling Pathways in Doxorubicin Sensitive and Methotrexate Resistant PLC/
Nasrin Shojaie1, Seyed Mahmood Ghaffari1
1Biochemistry Group, Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran.
Objective:
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.
Materials And Methods:
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).
Results:
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.
Conclusion:
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.
Insights
Multi-drug resistance in hepatocellular carcinoma involves altered Wnt and NF-κB pathways. Chemotherapy drugs impact these pathways, affecting cancer stem cell maintenance and apoptosis.
Area of Science:
- Oncology
- Molecular Biology
- Cancer Research
Background:
- Multi-drug resistance (MDR) is a significant challenge in treating aggressive cancers like hepatocellular carcinoma (HCC).
- Aberrant activation of signaling pathways, including Wnt and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), is implicated in MDR and cancer stem cell (CSC) maintenance.
- Understanding pathway alterations is crucial for developing effective chemo-resistant cancer therapies.
Purpose of the Study:
- To investigate alterations in Wnt and NF-κB signaling pathways in chemotherapy-resistant hepatocellular carcinoma cells.
- To assess key molecular parameters associated with these pathways in the context of drug resistance.
Main Methods:
- Utilized methylthiazol tetrazolium (MTT) assay, acridine orange/ethidium bromide (AO/EtBr) staining, Hoechst 33342 staining, DNA fragmentation, and colony formation assays.
- Investigated cytotoxic effects of methotrexate (MTX) and doxorubicin (DOX) on PLC/PRF/5 hepatocellular carcinoma cells.
- Employed semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) to analyze the expression of 11 genes involved in MDR.
Main Results:
- PLC/PRF/5 cells showed sensitivity to DOX and resistance to MTX.
- RT-PCR revealed significant downregulation of β-catenin and overexpression of ABCG2 in DOX- and MTX-treated cells.
- Hypoxia-inducible factor 1-alpha (HIF-1α) and p65 (NF-κB subunit) were notably expressed in MTX-resistant cells.
Conclusions:
- Anti-cancer drugs can target multiple cellular pathways, including Wnt and NF-κB signaling.
- Chemotherapy resistance in hepatocellular carcinoma is associated with alterations in these key pathways.
- HIF-1α was identified as a significantly induced anti-apoptotic protein in chemoresistant cells.
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