Yeast-Based Screen to Identify Natural Compounds with a Potential Therapeutic Effect in Hailey-Hailey Disease
"> Figure 1
<p>(<b>A</b>) General flowchart of the natural products screen approach. The primary screening is performed to identify compounds that alleviate the oxidative stress of <span class="html-italic">pmr1</span>-mutant cells. Then the effects of the positive hits are tested for the other phenotypes of the mutant strain. The final step is to test the selected molecules on the human cell cultures used as model for Hailey-Hailey disease; (<b>B</b>) The <span class="html-italic">PMR1</span>-deleted strain exposed or not for 24 h to different natural products was tested for its ability to grow with or without the 60 µM menadione or 4 mM H<sub>2</sub>O<sub>2</sub>. Wild type cells (WT) were used as control.</p> "> Figure 2
<p>Analysis of calcium alteration. WT and <span class="html-italic">Klpmr1∆</span> cells exposed or not to the individual natural molecules, were grown for 24 h in Yeast Extract-Peptone-Dextrose (YPD) medium at 30 °C. Then, serial dilutions of the cultures were spotted onto solid medium supplemented or not with 20 mM EGTA. Scale bar:</p> "> Figure 3
<p>Chitin distribution of mutant cells treated with the six selected products. <span class="html-italic">PMR1</span>-deleted cells, grown with or without the individual compounds for 24 h at 30 °C, were stained with the chitin-binding dye CFW. At least 500 cells were analyzed for each treatment to determine the percentage of cell wall recovery. Wild type cells (WT) represent the positive control. Scale bar 2 µm.</p> "> Figure 4
<p>Effect of the natural compounds on the altered mitochondrial function of <span class="html-italic">pmr1∆</span> cells. The mutant cells, untreated or treated with the indicated molecules for 24 h, were stained with the vital dye DASPMI and immediately the fluorescence micrographs were taken. To calculate the percentage of cells with altered tubular mitochondria morphology, at least 500 cells were analyzed for each condition. Wild type strain (WT) was used as a control. Scale bar 2 µm.</p> "> Figure 5
<p>Keratinocytes-derived cell line, HaCaT, was transfected with either siRNA-CTR or siRNA-ATP2C1; 24 h post-transfection, cells were treated with the indicated compounds at 10 µM for a further 24 h and analyzed by microscopy. (100× magnification). The potencies (EC<sub>50</sub> = 0.8 µM +/− 0.1) of Kaempferol were obtained from the dose–response curves using GraphPad Prism (GraphPad Software, La Jolla, CA, USA). Scale bar: 50 µm.</p> "> Figure 6
<p>(<b>A</b>,<b>B</b>) NHKCs (primary human keratinocytes) were transfected with control (siRNA-CTR) or ATP2C1-specific siRNA oligonucleotides; 24 h later, cells were treated with Kaempferol (10 µΜ) for 24 h and analyzed by microscopy. (100× magnification). Each of the lower images is an enlarged subset of the image above. Scale bar: 50 µm.</p> "> Figure 7
<p>Cell extracts were prepared from both NHKCs (<b>A</b>,<b>B</b>) and HaCaT cells (<b>C</b>,<b>D</b>) transfected with either control (siRNA-CTR) or ATP2C1-specific siRNA oligonucleotides; 24 h later, cells were treated with Kaempferol (10 µΜ) for 24 h and the cell extracts analyzed by western blot; (<b>E</b>) Cells were treated as in C, and expression of NRF2 was determined by RT-PCR; (<b>F</b>) Keratinocytes-derived cell line, HaCaT, was transfected with either siRNA-CTR or siRNA-ATP2C1 and cells were analyzed by flow cytometry. The percentage of ROS-positive cells is also shown. The absolute value of ROS of both from siRNA-CTR and siRNA-ATP2C1 Kaempferol-untreated cells was arbitrary indicated as 100%.</p> "> Figure 8
<p>(<b>A</b>–<b>C</b>) NHKC cells were transfected with control (siRNA-CTR) or ATP2C1-specific siRNA oligonucleotides; 24 h later, cells were treated with Kaempferol (10 µΜ) for 24 h, the total RNA extracted, and the expression of the indicated targets analyzed by RT-PCR; (<b>D</b>) ATP production in both yeast (left) and primary human keratinocytes (right). ATP levels were analyzed in <span class="html-italic">Klpmr1∆ and WT</span> cells, and ATP production was assessed in primary human keratinocytes transfected with control (siRNA-CTR) or ATP2C1-specific siRNA oligonucleotides.</p> ">
Abstract
:1. Introduction
2. Results
2.1. Primary Screen of Chemical Libraries Using KLPMR1-Based Assay
2.2. Yeast-Hits Rescue Multiple Defects in pmr1Δ Cells
2.3. Cell Wall Phenotype
2.4. Mitochondrial Morphology
2.5. Drugs Active in the Yeast-Based Assay Were Also Active in Human Cells
2.6. Potential Mechanism of Kaempferol against ATP2C1-Induced Oxidative-Stress through Regulation of Nuclear Factor Erythroid-2-Related Factor 2 Signaling
2.7. Increased Mitochondrial Activity as a Source of Oxidative Stress in ATP2C1-Defective Keratinocytes
3. Discussion
4. Materials and Methods
4.1. Yeast Strains, Growth Conditions
4.2. Library Screen
4.3. Fluorescence Microscopy
4.4. Primary Human Keratinocytes
4.5. Cell Culture and Transfection
4.6. Reagents and Immunoblotting
4.7. RNA Analysis and Reverse Transcriptase-Polymerase Chain Reaction
5. Conclusions
Supplementary Materials
Author Contributions
Acknowledgments
Conflicts of Interest
References
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Chemicals | Oxidative Stress Selection | EC50 (mM) |
---|---|---|
S1973 (Cyclocytidine) | H2O2 | 51.26 ± 1.19 |
S2267 (Azomycin) | Menadione | 102.64 ± 0.50 |
S2314 (Kaempferol) | H2O2 | 50.5 ± 0.44 |
S2328 (Nalidixic acid) | Menadione | 50.13 ± 4.84 |
S2386 (Indirubin) | Menadione | 48.56 ± 3.01 |
S2387 (Lappaconite) | Menadione | 9.35 ± 0.42 |
SyBr Green Assays | Sequence 5′-3′ |
GST-M1 Fw | AGAGGAGAAGATTCGTGTGG |
GST-M1 Rev | TGTTTCCTGCAAACCATGGC |
GAPDH Fw | TGCACCACCAACTGCTTAG |
GAPDH Rev | GAGGCAGGGATGATGTTC |
Taqman Gene Expression Assays | Assay Reference Number |
NFE2L2 (NRF2) | Hs00975961_g1 |
GAPDH | Hs99999905_m1 |
NQO1 | Hs02512143_s1 |
SOD1 | Hs00533490_m1 |
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Ficociello, G.; Zonfrilli, A.; Cialfi, S.; Talora, C.; Uccelletti, D. Yeast-Based Screen to Identify Natural Compounds with a Potential Therapeutic Effect in Hailey-Hailey Disease. Int. J. Mol. Sci. 2018, 19, 1814. https://doi.org/10.3390/ijms19061814
Ficociello G, Zonfrilli A, Cialfi S, Talora C, Uccelletti D. Yeast-Based Screen to Identify Natural Compounds with a Potential Therapeutic Effect in Hailey-Hailey Disease. International Journal of Molecular Sciences. 2018; 19(6):1814. https://doi.org/10.3390/ijms19061814
Chicago/Turabian StyleFicociello, Graziella, Azzurra Zonfrilli, Samantha Cialfi, Claudio Talora, and Daniela Uccelletti. 2018. "Yeast-Based Screen to Identify Natural Compounds with a Potential Therapeutic Effect in Hailey-Hailey Disease" International Journal of Molecular Sciences 19, no. 6: 1814. https://doi.org/10.3390/ijms19061814