A combination of AZD5363 and FH5363 induces lethal autophagy in transformed hepatocytes
Tapas Patra1, Keith Meyer2, Ratna B Ray3
1Department of Internal Medicine, Saint Louis University, Saint Louis, MO, 63104, USA. tapas.patra@health.slu.edu.
Abstract:
Hepatocellular carcinoma (HCC) is one of the major causes of cancer-related death worldwide. High Akt activation and aberrant β-catenin expression contribute to HCC cell proliferation, stem cell generation, and metastasis. Several signaling pathway-specific inhibitors are in clinical trials and display different efficacies against HCC. In this study, we observed that a β-catenin inhibitor (FH535) displays antiproliferative effect on transformed human hepatocytes (THH). A combination treatment of these cells with FH535 and Akt inhibitor (AZD5363) exerted a stronger effect on cell death. Treatment of THH with AZD5363 and FH535 inhibited cell-cycle progression, enhanced autophagy marker protein expression, and autophagy-associated death, while FH535 treatment alone induced apoptosis. The use of chloroquine or z-VAD further verified these observations. Autophagy flux was evident from lowering marker proteins LAMP2, LAPTM4B, and autophagic protein expression by confocal microscopy using mCherry-EGFP-LC3 reporter construct. A combination treatment with AZD5363 and FH535 enhanced p53 expression, by modulating MDM2 activation; however, AZD5363 treatment alone restricted p53 to the nucleus by inhibiting dynamin-related protein activation. Nuclear p53 plays a crucial role for activation of autophagy by regulating the AMPK-mTOR-ULK1 pathway. Hep3B cells with null p53 did not modulate autophagy-dependent death from combination treatment. Together, our results strongly suggested that a combination treatment of Akt and β-catenin inhibitors exhibits efficient therapeutic potential for HCC.
Insights
Combining Akt and β-catenin inhibitors, FH535 and AZD5363, shows promise for hepatocellular carcinoma (HCC) treatment. This combination enhances cell death and autophagy, offering a potential new therapy for HCC.
Area of Science:
- Oncology
- Molecular Biology
- Cellular Biology
Background:
- Hepatocellular carcinoma (HCC) is a leading cause of cancer mortality globally.
- Akt activation and β-catenin signaling drive HCC progression, proliferation, and metastasis.
- Targeted inhibitors are being investigated for HCC treatment efficacy.
Purpose of the Study:
- To evaluate the antiproliferative and cell death-inducing effects of a β-catenin inhibitor (FH535) and an Akt inhibitor (AZD5363) on transformed human hepatocytes (THH).
- To investigate the mechanisms underlying cell death, including apoptosis and autophagy, induced by these inhibitors, alone and in combination.
- To explore the role of p53 in mediating the combined therapeutic effects.
Main Methods:
- Treatment of THH with FH535 and/or AZD5363.
- Assessment of cell-cycle progression, apoptosis, and autophagy.
- Confocal microscopy using mCherry-EGFP-LC3 reporter for autophagy flux analysis.
- Western blotting to analyze protein expression (e.g., p53, MDM2, dynamin-related protein).
- Experiments with p53-null Hep3B cells.
Main Results:
- FH535 exhibited antiproliferative effects on THH.
- Combination treatment with FH535 and AZD5363 enhanced cell death compared to single-agent treatment.
- Combined treatment induced cell-cycle arrest and promoted autophagy-associated death, while FH535 alone induced apoptosis.
- Autophagy flux was confirmed via reporter assays and marker protein analysis.
- Combination treatment upregulated p53 expression by modulating MDM2, whereas AZD5363 alone inhibited p53 nuclear translocation.
- p53-null cells did not exhibit autophagy-dependent death upon combination treatment, highlighting p53's critical role.
Conclusions:
- Combination therapy with Akt and β-catenin inhibitors demonstrates significant therapeutic potential for HCC.
- The synergistic effect involves enhanced cell death through apoptosis and p53-mediated autophagy.
- Targeting both Akt and β-catenin pathways offers a promising strategy for HCC treatment.
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