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Published on: December 5, 2020
Antitumour activity of 3-nitropropionic acid from Phomopsis sp. and optimization of fermentation conditions
1Animal Disease Prevention and Food Safety Key Laboratory of Sichuan Province, Key Laboratory of Bio-Resources and Eco-Environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu, Sichuan, China.
Unlabelled:
In this study, 3-nitropropionic acid (3-NPA) was separated and purified from endophytic fungi belonging to Phomopsis sp. and its cytotoxicity was determined by MTT assay. Treatment with 3-NPA for 24 h resulted in a dose-dependent apoptosis in MCF-7 cells. Through quantitative detection of the genes that are closely related to the Bcl-2 signalling pathway, there was an increased expression of p53 and Bax and a decreased expression of Bcl-2, which indicated apoptosis in these cells. Meanwhile, the overexpression of PARA (poly ADP-ribose polymerase) and apoptosis inducing factor (AIF) also suggested that 3-NPA induced cellular apoptosis through a caspase-3-independent pathway in caspase-3-deficient MCF-7 cells. The fermentation condition was also improved to produce more 3-NPA: glucose as a carbon source and yeast extract as a nitrogen source, fermentation for 8 days at 32°C and a solution environment of pH 5·0. Under these conditions, the yield of 3-NPA was increased to 529 mg l(-1) compared with 410 mg l(-1) under traditional fermentation conditions.
Significance And Impact Of The Study:
3-Nitropropionic acid is a mitochondrial inhibitor and has some useful bioactivities such as antibacterial activity. In this paper we found that 3-NPA also has obvious cytotoxicity, so we studied its antitumour activity and tried to determine the antitumour molecular mechanism, opening a new perspective for potential antitumour prodrug development. As 3-NPA is often obtained from natural products with a low yield, in order to overcome the disadvantage of an endophytic fungi source of 3-NPA, we optimized the fermentation conditions for 3-NPA in Phomopsis sp. to obtain the maximum production of 3-NPA.
Insights
3-Nitropropionic acid (3-NPA) isolated from Phomopsis sp. induces apoptosis in cancer cells via a caspase-3-independent pathway. Optimized fermentation conditions significantly increased 3-NPA production for potential anticancer drug development.
Area of Science:
- Biochemistry
- Molecular Biology
- Natural Product Chemistry
Background:
- 3-Nitropropionic acid (3-NPA) is known as a mitochondrial inhibitor with various bioactivities.
- Endophytic fungi are a source of novel bioactive compounds, including 3-NPA.
- Understanding the cytotoxic mechanisms of natural products is crucial for drug development.
Purpose of the Study:
- To isolate and purify 3-NPA from Phomopsis sp. endophytic fungi.
- To investigate the cytotoxic and antitumour activity of 3-NPA on MCF-7 cells.
- To elucidate the molecular mechanism of 3-NPA-induced apoptosis.
- To optimize fermentation conditions for enhanced 3-NPA production.
Main Methods:
- Isolation and purification of 3-NPA from Phomopsis sp.
- Cytotoxicity assessment using MTT assay.
- Apoptosis detection via gene expression analysis (Bcl-2 signaling pathway, p53, Bax, Bcl-2).
- Analysis of apoptosis-related proteins (PARA, AIF) and caspase-3 activity.
- Optimization of fermentation parameters (carbon/nitrogen sources, temperature, pH, duration).
Main Results:
- 3-NPA was successfully isolated and purified from Phomopsis sp.
- 3-NPA demonstrated dose-dependent cytotoxicity and induced apoptosis in MCF-7 cells.
- Gene expression analysis revealed upregulation of p53 and Bax, and downregulation of Bcl-2.
- Overexpression of PARA and AIF indicated a caspase-3-independent apoptotic pathway.
- Optimized fermentation conditions increased 3-NPA yield to 529 mg/L.
Conclusions:
- 3-NPA exhibits significant antitumour activity by inducing apoptosis through a caspase-3-independent mechanism.
- The study provides a foundation for developing 3-NPA as a potential anticancer prodrug.
- Optimized fermentation offers a viable strategy to overcome low yield limitations from natural sources.
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