PHNQ from Evechinus chloroticus Sea Urchin Supplemented with Calcium Promotes Mineralization in Saos-2 Human Bone Cell Line
Abstract
:1. Introduction
2. Results
2.1. Extraction of PHNQs from E. choloticus Spine
2.2. Cytotoxic Activity of E. chloroticus PHNQ Extract
2.3. The Effect of CaCl2, E. choloroticus PHNQ Extract and Echinochrome A on the Proliferation of Saos-2 Cells
2.4. Effect of Adding CaCl2 with Echinochrome A and E. choloroticus PHNQ Extract on Cell Viability
2.5. Effect of CaCl2 on Minerlization of Saos-2 Cells and the Mineralization Assay Evaluation
2.6. Effect of PHNQ Extract and Echinochrome A on Mineralization of Saos-2 Cells
2.7. Effect of PHNQ on Mineralization in the Presence of CaCl2
3. Discussion
4. Materials and Methods
4.1. The Source of Sea Urchin Spine Samples and Echinochrome A Standard
4.2. Preparation of Sea Urchin Spine Crude Extract
4.3. Characterisation of PHNQ Compounds Using HPLC with Diode-Array Detection and Mass Spectrometry (HPLC-DAD/MS)
4.4. Determination of Cytotoxicity of PHNQ by MTT Assay Using Saos-2 Cells (48 h Incubation Time)
4.5. Determination of Cytotoxicity of PHNQ, Echinochrome A, CaCl2, and PHNQ or Echinochrome A Supplemented with CaCl2 by MTT Assay Using Saos-2 Cells (21 days Incubation Time)
4.6. Determination of Mineralized Nodule Formation by Xylenol Orange Staining
4.7. Determination of Mineralized Nodule Formation by von Kossa Staining
4.8. Statistical Analysis
5. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Appendix A
Appendix B
Appendix B.1. PHNQ Stability in Cell Culture Media
Appendix B.2. Results of PHNQ Stability in Cell Culture Media
CaCl2 (mM)/PHNQ Concentration (µg/mL) | 15.625 (Low) | 31.25 (Medium) | 62.5 (High) |
---|---|---|---|
Ca 0 mM | 73.86 ± 14.98 a | 67.21 ± 10.98 b | 84.38 ± 4.93 b |
Ca 0.5 mM | 77.93 ± 17.65 a | 80.90 ± 10.71 ab | 71.46 ± 3.26 ab |
Ca 1.0 mM | 69.99 ± 8.80 a | 73.67 ± 3.71 ab | 74.91 ± 3.46 b |
Ca 1.5 mM | 68.33 ± 19.98 a | 68.66 ± 8.92 b | 57.13 ± 5.27 b |
Ca 2.0 mM | 66.30 ± 22.70 a | 68.00 ± 7.99 b | 80.85 ± 7.68 b |
Ca 2.5 mM | 66.19 ± 16.93 a | 70.00 ± 10.38 ab | 63.87 ± 8.10 b |
Ca 3.0 mM | 65.41 ± 16.98 a | 68.26 ± 13.20 b | 86.25 ± 13.73 b |
vehicle control ** | 103.2 ± 5.05 a | 106.77 ± 3.19 a | 106.24 ± 8.03 a |
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Sample Name | Final Concentration in Media |
---|---|
CaCl2 control | CaCl2 0–4.0 mM |
High concentration of echinochrome A | 62.5 µg/mL |
Medium concentration of echinochrome A | 31.25 µg/mL |
Low concentration of echinochrome A | 15.625 µg/mL |
High concentration of PHNQ | 62.5 µg/mL |
Medium concentration of PHNQ | 31.25 µg/mL |
Low concentration of PHNQ | 15.625 µg/mL |
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Hou, Y.; Carne, A.; McConnell, M.; Mros, S.; Vasileva, E.A.; Mishchenko, N.P.; Burrow, K.; Wang, K.; Bekhit, A.A.; Bekhit, A.E.-D.A. PHNQ from Evechinus chloroticus Sea Urchin Supplemented with Calcium Promotes Mineralization in Saos-2 Human Bone Cell Line. Mar. Drugs 2020, 18, 373. https://doi.org/10.3390/md18070373
Hou Y, Carne A, McConnell M, Mros S, Vasileva EA, Mishchenko NP, Burrow K, Wang K, Bekhit AA, Bekhit AE-DA. PHNQ from Evechinus chloroticus Sea Urchin Supplemented with Calcium Promotes Mineralization in Saos-2 Human Bone Cell Line. Marine Drugs. 2020; 18(7):373. https://doi.org/10.3390/md18070373
Chicago/Turabian StyleHou, Yakun, Alan Carne, Michelle McConnell, Sonya Mros, Elena A. Vasileva, Natalia P. Mishchenko, Keegan Burrow, Ke Wang, Adnan A. Bekhit, and Alaa El-Din A. Bekhit. 2020. "PHNQ from Evechinus chloroticus Sea Urchin Supplemented with Calcium Promotes Mineralization in Saos-2 Human Bone Cell Line" Marine Drugs 18, no. 7: 373. https://doi.org/10.3390/md18070373
APA StyleHou, Y., Carne, A., McConnell, M., Mros, S., Vasileva, E. A., Mishchenko, N. P., Burrow, K., Wang, K., Bekhit, A. A., & Bekhit, A. E. -D. A. (2020). PHNQ from Evechinus chloroticus Sea Urchin Supplemented with Calcium Promotes Mineralization in Saos-2 Human Bone Cell Line. Marine Drugs, 18(7), 373. https://doi.org/10.3390/md18070373