NF-κB/RELA Knockout Reveals a Role in Expression of HIF-1α and NES in A172 Cell Line: Suggestion Ecteinascidin-743 as a Suitable Drug
Main Article Content
Keywords
Glioblastoma multiforme; NF-κB; RELA; CRISPR/Cas9; Ecteinascidin-743
Abstract
The NF-κB signalling pathway is one of the most critical controllers of the inflammatory response in glioblastoma multiforme (GBM). In this study, the knockout association of the RELA subunit was examined by expressing some genes connected tothe nuclear factor κB (NF-κB) pathway in sustaining hypoxic circumstances and GBM stem cells (GSCs) properties. A suitable drug is also suggested to target RELA . Targeted deletion of the RELAsubunit in human GBM cells (A172) was performed with CRISP R/Cas9. Nestin (NES) and hypoxia inducible factor 1 alpha (HIF-1α) genes expression levels and induction of apoptosis in transfected and control cells were assessed by Real-time PCR and flow cytometry, respectively. Using the Auto Dock Vina software, a molecular docking study was conducted to find the optimum intermolecular interaction between RELA protein and four potential drugs. Real-time PCR results showed a decrease in the expression of HIF-1α and NES genes in the transfected cell population compared to the control cells (p< 0.0102, p< 0.0012, and P< 0.0442, respectively). Flow cytometry results showed a significantly increased induction of apoptosis in the transfected cells compared to the control cell population. The results of docking revealed that Ecteinascidin-743 has the best intermolecular interaction with RELAprotein. In conclusion, the RELA subunit seems to be one of the factors affecting hypoxia, apoptosis, and change in stemness genes expression levels in GBM. Therefore, it is recommended to knock out the NF-κB signalling pathway or to use Ecteinascidin-743 in future studies.
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designing antiviral drugs against dengue fever via targeting host-based approaches. Microorganisms 7, 296.
3. Broekman, M.L., Maas, S.L.N., Abels, E.R., Mempel, T.R., Krichevsky, A.M., Breakefield, X.O., 2018. Multidimensional communication in
the microenvirons of glioblastoma. Nat. Rev. Neurol. 14, 482–495.
4. Cahill, K.E., Morshed, R.A., Yamini, B., 2015. Nuclear factor-κB in glioblastoma: insights into regulators and targeted therapy. Neuro. Oncol. 18, 329–339.
5. Filatova, A., Acker, T., Garvalov, B.K., 2013. The cancer stem cell niche (s): the crosstalk between glioma stem cells and their microenvironment. Biochim. Biophys. Acta (BBA)-General Subj. 1830, 2496–2508.
6. Friedmann-Morvinski, D., Narasimamurthy, R., Xia, Y., Myskiw, C., Soda, Y., Verma, I.M., 2016. Targeting NF-κB in glioblastoma: A therapeutic
approach. Sci. Adv. 2, e1501292.
7. Germano, G., Frapolli, R., Belgiovine, C., Anselmo, A., Pesce, S., Liguori, M., Erba, E., Uboldi, S., Zucchetti, M., Pasqualini, F., 2013. Role of
macrophage targeting in the antitumor activity of trabectedin. Cancer Cell 23, 249–262.
8. Germano, G., Frapolli, R., Simone, M., Tavecchio, M., Erba, E., Pesce, S., Pasqualini, F., Grosso, F., Sanfilippo, R., Casali, P.G., 2010. Antitumor and anti-inflammatory effects of trabectedin on human myxoid liposarcoma cells. Cancer Res. 70, 2235–2244.
9. Gilmore, T.D., 2021. NF-κB and human cancer: what have we learned over the past 35 years? Biomedicines 9, 889.
10. Gimple, R.C., Bhargava, S., Dixit, D., Rich, J.N., 2019. Glioblastoma stem cells: lessons from the tumor hierarchy in a lethal cancer. Genes Dev. 33, 591–609.
11. Hoesel, B., Schmid, J.A., 2013. The complexity of NF-κB signaling in inflammation and cancer. Mol. Cancer 12, 1–15.
12. Ishiwata, T., Teduka, K., Yamamoto, T., Kawahara, K., Matsuda, Y., Naito, Z., 2011. Neuroepithelial stem cell marker nestin regulates the migration, invasion and growth of human gliomas. Oncol. Rep. 26, 91–99.
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14. Kaltschmidt, C., Banz-Jansen, C., Benhidjeb, T., Beshay, M., Förster, C., Greiner, J., Hamelmann, E., Jorch, N., Mertzlufft, F., Pfitzenmaier, J., 2019. A role for NF-κB in organ specific cancer and cancer stem cells. Cancers (Basel). 11, 655.
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compound databases. Nucleic Acids Res. 44, D1202–D1213.
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18. Lawrence, T., 2010. Macrophages and NF-κB in cancer. NF-kB Heal. Dis. 171–184.
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21. Miller, S.C., Huang, R., Sakamuru, S., Shukla, S.J., Attene-Ramos, M.S., Shinn, P., Van Leer, D., Leister, W., Austin, C.P., Xia, M., 2010. Identification of known drugs that act as inhibitors of NF-κB signaling and their mechanism of action. Biochem. Pharmacol. 79, 1272–1280.
22. Nogueira, L., Ruiz-Ontanon, P., Vazquez-Barquero, A., Lafarga, M., Berciano, M.T., Aldaz, B., Grande, L., Casafont, I., Segura, V., Robles, E.F., 2011. Blockade of the NFκB pathway drives differentiating glioblastoma-initiating cells into senescence both in vitro and in vivo. Oncogene 30,
3537–3548.
23. Papale, M., Buccarelli, M., Mollinari, C., Russo, M.A., Pallini, R., Ricci-Vitiani, L., Tafani, M., 2020. Hypoxia, inflammation and necrosis as
determinants of glioblastoma cancer stem cells progression. Int. J. Mol. Sci. 21, 2660.
24. Parvizpour, S., Masoudi-Sobhanzadeh, Y., Pourseif, M.M., Barzegari, A., Razmara, J., Omidi, Y., 2021. Pharmacoinformatics-based phytochemical screening for anticancer impacts of yellow sweet clover, Melilotus officinalis (Linn.) Pall. Comput. Biol. Med. 138, 104921.
25. Pesenti, C., Navone, S.E., Guarnaccia, L., Terrasi, A., Costanza, J., Silipigni, R., Guarneri, S., Fusco, N., Fontana, L., Locatelli, M., 2019. The genetic landscape of human glioblastoma and matched primary cancer stem cells reveals intratumour similarity and intertumour heterogeneity. Stem Cells Int. 2019.
26. Rinkenbaugh, A., Cogswell, P., Calamini, B., Dunn, D., Persson, A., Weiss, W., Lo, D., Baldwin, A., 2016. IKK/NF-?B signaling contributes to
glioblastoma stem cell maintenance. Oncotarget 7. https://doi.org/10.18632/oncotarget.12507
27. Saranaruk, P., Kariya, R., Sittithumcharee, G., Boueroy, P., Boonmars, T., Sawanyawisuth, K., Wongkham, C., Wongkham, S., Okada, S.,
Vaeteewoottacharn, K., 2020. Chromomycin A3 suppresses cholangiocarcinoma growth by induction of S phase cell cycle arrest and
suppression of Sp1‑related anti‑apoptotic proteins. Int. J. Mol. Med. 45, 1005–1016.
28. Sharma, A., Shiras, A., 2016. Cancer stem cellvascular endothelial cell interactions in glioblastoma. Biochem. Biophys. Res. Commun.
473, 688–692.
29. Soubannier, V., Stifani, S., 2017. NF-κB Signalling in Glioblastoma. Biomedicines 5, 29. https://doi.org/10.3390/biomedicines5020029
30. Su, Z., Han, S., Jin, Q., Zhou, N., Lu, Junwan, Shangguan, F., Yu, S., Liu, Y., Wang, L., Lu, Jianglong, 2021. Ciclopirox and bortezomib
synergistically inhibits glioblastoma multiforme growth via simultaneously enhancing JNK/p38 MAPK and NF-κB signaling. Cell Death Dis. 12, 1–13.
31. Tafani, M., Di Vito, M., Frati, A., Pellegrini, L., De Santis, E., Sette, G., Eramo, A., Sale, P., Mari, E., Santoro, A., 2011. Pro-inflammatory gene
expression in solid glioblastoma microenvironment and in hypoxic stem cells from human glioblastoma. J. Neuroinflammation 8, 1–16.
32. Tan, C.R.C., Abdul-Majeed, S., Cael, B., Barta, S.K., 2019. Clinical pharmacokinetics and pharmacodynamics of bortezomib. Clin.
Pharmacokinet. 58, 157–168.
33. Trott, O., Olson, A.J., 2010. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J. Comput. Chem. 31, 455–461.
34. Zhang, Q., Feng, Y., Kennedy, D., 2017. Multidrugresistant cancer cells and cancer stem cells hijack cellular systems to circumvent systemic therapies, can natural products reverse this? Cell. Mol. Life Sci. 74, 777–801.
35. Zhu, G., Zhao, M., Han, Q., Tan, Y., Sun, Y.U., Bouvet, M., Singh, S.R., Ye, J., Hoffman, R.M., 2019. Combination of trabectedin with oxaliplatinum and 5-fluorouracil arrests a primary colorectal cancer in a patient-derived orthotopic xenograft mouse model. Anticancer Res. 39, 5999–6005.
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Feb 6, 2023
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Sima Salmani, Dhiya Altememy, Sepideh Parvizpoor, Akram Alizadeh, & Sorayya Ghasemi. (2023). NF-κB/RELA Knockout Reveals a Role in Expression of HIF-1α and NES in A172 Cell Line: Suggestion Ecteinascidin-743 as a Suitable Drug. Journal of Population Therapeutics and Clinical Pharmacology, 30(1), 206–217. https://doi.org/10.47750/jptcp.2023.1041
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