Project: "Proteomic approach to recognition of targets of antineoplastic activity of AMPK-induced agents in breast cancer MCF-7 cells"
Principal Investigator: Thi Yen Ly Huynh
Co-Investigator: Ilona Zaręba, PhD
Principal Investigator’s Supervisor: Professor Jerzy Palka
Department: Department of Medicinal Chemistry (Faculty of Pharmacy)
- Research project objectives/Research hypothesis
Rapid accumulation of proline concentration has been observed in various cancers. It is probably due to the degradation of collagen type I in the extracellular matrix by collagenases and intracellularly by prolidase which releases proline from imidodipeptides. An increase in prolidase activity may boost proline concentration in the cytoplasm but, as previously found, it is recycled for collagen re-synthesis. Thus, the enzyme plays an important role in the regulation of intracellular proline concentration and collagen biosynthesis.
Cytoplasmic proline level is apparently affected by proline utilizing processes, namely degradation of proline by proline dehydrogenase/proline oxidase (PRODH/POX) in mitochondria and utilization of this amino acid for collagen biosynthesis. Proline oxidase is a flavin-dependent enzyme associated with the inner mitochondrial membrane, enable to converse proline into ∆1-pyrroline-5-carboxylate (P5C) with the release of either ATP for survival or reactive oxygen species (ROS) for apoptosis/autophagy. However, the mechanism that switches POX from tumour growth inhibiting to growth supporting factor is unknown.
In such conditions, accumulation of proline concentration in the cytoplasm was accompanied by utilization of proline for collagen biosynthesis or for production of P5C via activation of POX based on energy status of the cell and regulation of AMP kinase (AMPK) which is activated by phosphorylation when the AMP/ATP ratio rises. AMPK is a critical factor, which mediates adaptation for such metabolic changes.
One of the energy providing substrates is proline, which can be recovered from collagen degradation products as well as derived from α-ketoglutarate, glutamic acid, glutamine, and ornithine.
Therefore, it is postulated that complex regulation of prolidase activity/PRODH/POX/collagen biosynthesis and amino acids metabolism by environmental factors may represent an interface that triggers apoptosis or survival mode in both normal and cancer cells.
Metformin is a worldwide prescribed drug applied for treatment of type II diabetes. It enhances cell sensitivity to insulin, inhibits mitochondrial respiratory chain (complex I) and has recently been proposed as inducer of AMP-activated protein kinase (AMPK).
We have therefore hypothesized that induction of AMPK can stimulate apoptosis in tumour cells by a cascade of processes involving up-regulation of PRODH/POX expression and ROS production under proline availability. The purpose of this project is to build a case for the presented hypothesis and to evaluate the effect of AMPK activation on the signalling pathways and amino acids profile involved in apoptosis/autophagy regulation in wild type breast cancer MCF-7 cells and the cell line with knock-down PRODH/POX.
- Research project methodology
The impact of AMPK-inducing agents on cell viability, DNA biosynthesis, cell growth, collagen biosynthesis, prolidase activity and PRODH/POX expression are evaluated in wild-type MCF-7 cells and PRODH/POX-knock down MCF-7 cells as well as the effect of AMPK-inducing agents on expression of apoptosis/autophagy markers in both cell lines are analysed by Western blot and immunocytochemistry. Profiles of selected amino acids in these cells treated with AMPK-inducing agents are evaluated by LCMS.
- Expected impact of the research project on the development of science, civilization and the society
This project aims to clarify the molecular mechanism of anti-cancer action of AMPK-inducing agents (e.g. metformin, thiazolidinothiones) in the experimental model of breast cancer cells. The biological effects of AMPK-inducing agents on breast cancer cells will be assessed in the perspective of potential use in development of new targeted cancer pharmacotherapy. Knowing the target of antineoplastic action of these agents allows to improve the pharmacotherapy of cancer.