Ovcar-3 Ovarian Adenokarsinoma Hücre Hattında SVIP ve UPR Proteinlerinin İmmünoekspresyonunun Araştırılması

Yıl 2024, , 299 - 304, 08.10.2024

Ebru Alimoğulları , Bahar Kartal

https://doi.org/10.32708/uutfd.1524387

Öz

En ölümcül jinekolojik kanser yumurtalık kanseridir. Hayati bir hücre organeli olan endoplazmik retikulum (ER), çok çeşitli çözünür ve çözünmez proteinlerin katlanması, sentezi ve modifikasyonunda rol oynar. ER stresi, evrimsel süreçte korunmuş bir hücre stres mekanizması olan katlanmamış protein yanıtını (UPR) başlatır. UPR’a, üç ER transmembran sensörü aracılık eder: IRE1, ATF6 ve PERK. ERAD inhibitörü küçük VCP/p97 ile etkileşen proteindir (SVIP). Çalışmanın amacı, Ovcar-3 insan ovarian adenokarsinoma hücre hattında SVIP ile ER stres protein belirteçleri arasındaki ilişkiyi araştırmaktır. SVIP ve GRP78, PERK, ATF4 immünoekspresyon düzeyleri analiz edildi. Ayrıca, immünofloresan kullanılarak, üç ER sensörünün ve SVIP'in kolokalizasyonu belirlendi. SVIP ve GRP78, ATF4 ve PERK'nin immünoekspresyonu OVCAR-3 hücre hattında gösterildi. Ek olarak, immünofloresan sonuçları OVCAR-3 hücrelerinin sitoplazmasında SVIP ve UPR ile ilişkili proteinlerin kolokalizasyonunu gösterdi. Sonuç olarak, SVIP'nin hücresel lokalizasyonunu ve UPR yolunda yer alan proteinleri gösterdik ancak kanser hücrelerinde SVIP ile bu proteinler arasındaki ilişkiyi belirlemek için daha fazla çalışmaya ihtiyaç vardır.

Anahtar Kelimeler

Endoplazmik retikulum (ER) stresi, İmmünofloresan, Ovarian adenokarsinoma hücre hattı (OVCAR-3), Küçük VCP ile etkileşen protein (SVIP), Katlanmamış protein yanıtı (UPR)

Investigation of the Immunoexpression of SVIP and UPR Pathway Proteins in Ovarian Adenocarcinoma Cell Line OVCAR-3

Öz

Ovarian cancer is the deadliest gynecological cancer. The endoplasmic reticulum (ER), a vital cell organelle, is involved in the folding, synthesis, and modification of a wide range of soluble and insoluble proteins. ER stress initiates the unfolded protein response (UPR), an evolutionary conserved cell stress mechanism. The UPR is mediated by three ER transmembrane sensors: IRE1, ATF6, and PERK. An inhibitor of ERAD is a small VCP/p97-interacting protein (SVIP). The study aimed to investigate the relationship between SVIP and the ER stress protein markers in the human ovarian cancer cell line OVCAR-3. The SVIP and GRP78, PERK, ATF4 immunoexpression levels were analyzed. Furthermore, employing immunofluorescence, the colocalization of three ER sensors and SVIP was ascertained. The immunoexpression of SVIP and GRP78, ATF4, and PERK were shown in the OVCAR-3 cell line. Additionally, immunofluorescence results showed the colocalization of SVIP and UPR-related proteins in the cytoplasm of OVCAR-3 cells. In conclusion, we demonstrated the cellular localization of SVIP and the proteins involved in the UPR pathway. However, further studies are needed to determine the relation between SVIP and these proteins in cancer cells.

Anahtar Kelimeler

Endoplasmic reticulum (ER) stress, Immunofluorescence, Ovarian adenocarcinoma cell line (OVCAR-3), Small VCP-interacting protein (SVIP), Unfolded protein response (UPR)

Kaynakça

• 1. Kujawa KA, Lisowska KM. Rak jajnika--od biologii do kliniki [Ovarian cancer--from biology to clinic]. Postepy Hig Med Dosw (Online). 2015;69:1275-1290.
• 2. Hernandez L, Kim MK, Lyle LT, et al. Characterization of ovarian cancer cell lines as in vivo models for preclinical studies. Gynecol Oncol. 2016;142(2):332–40.
• 3. Zhang D, Li N, Xi Y, Zhao Y, Wang T. Diabetes mellitus and risk of ovarian cancer. A systematic review and meta-analysis of 15 cohort studies. Diabetes Res Clin Pract. 2017;130:43–52.
• 4. Yang L, Zhao Y, Wang Y, et al. The Effects of Psychological Stress on Depression. Curr Neuropharmacol. 2015;13(4):494–504.
• 5. Licaj I, Jacobsen BK, Selmer RM, et al. Smoking and risk of ovarian cancer by histological subtypes: an analysis among 300 000 Norwegian women. British journal of cancer. 2017;116(2):270–6.
• 6. Chen R, Alvero A, Silasi D, Steffensen K, Mor G. Cancers take their Toll—the function and regulation of Toll-like receptors in cancer cells. Oncogene. 2008;27(2):225–33.
• 7. Wang M, Kaufman RJ. Protein misfolding in the endoplasmic reticulum as a conduit to human disease. Nature. 2016;529(7586):326-35.
• 8. Bravo R, Parra V, Gatica D, et al. Endoplasmic reticulum and the unfolded protein response: dynamics and metabolic integration. Int Rev Cell Mol Biol. 2013;301:215-90.
• 9. Bertolotti A, Zhang Y, Hendershot LM, Harding HP, Ron D. Dynamic interaction of BiP and ER stress transducers in the unfolded-protein response. Nat Cell Biol. 2000 Jun;2(6):326-32.
• 10. Shen, J.; Chen, X.; Hendershot, L.; Prywes, R. ER stress regulation of ATF6 localization by dissociation of BiP/GRP78 binding and unmasking of Golgi localization signals. Dev. Cell 2002, 3, 99–111.
• 11. Sannino S, Brodsky JL. Targeting protein quality control pathways in breast cancer. BMC Biol. 2017 Nov 16;15(1):109.
• 12. Rozpedek W, Pytel D, Mucha B, et al. The Role of the PERK/eIF2α/ATF4/CHOP Signaling Pathway in Tumor Progression During Endoplasmic Reticulum Stress. Curr Mol Med. 2016;16(6):533-44.
• 13. Pakos-Zebrucka K, Koryga I, Mnich K, et al. The integrated stress response. EMBO Rep. 2016 Oct;17(10):1374-1395.
• 14. Ito D, Walker JR, Thompson CS, et al. Characterization of stanniocalcin 2, a novel target of the mammalian unfolded protein response with cytoprotective properties. Mol Cell Biol. 2004;24,9456-69.
• 15. Wu R, Zhang QH, Lu YJ, Ren K, Yi GH. Involvement of the IRE1α-XBP1 pathway and XBP1s-dependent transcriptional reprogramming in metabolic diseases. DNA Cell Biol. 2015 Jan;34(1):6-18.
• 16. Moenner M, Pluquet O, Bouchecareilh M, Chevet E. Integrated endoplasmic reticulum stress responses in cancer. Cancer Res. 2007;67(22):10631-4.
• 17. Guo F, Snapp EL. ERdj3 regulates BiP occupancy in living cells. Journal of cell science. 2013; 126(Pt 6):1429–39.
• 18. Hoseki J, Ushioda R, Nagata K. Mechanism and components of endoplasmic reticulum-associated degradation. Journal of biochemistry. 2010; 147(1):19–25.
• 19. Hiller MM, Finger A, Schweiger M, Wolf DH. ER degradation of a misfolded luminal protein by the cytosolic ubiquitin-proteasome pathway. Science. 1996; 273(5282):1725–8.
• 20. McCracken AA, Brodsky JL. Evolving questions and paradigm shifts in endoplasmic-reticulum-associated degradation (ERAD). BioEssays: news and reviews in molecular, cellular and developmental biology. 2003; 25(9):868–77.
• 21. Hampton RY. ER-associated degradation in protein quality control and cellular regulation. Current opin- ion in cell biology. 2002; 14(4):476–82.
• 22. Ye Y, Shibata Y, Yun C, Ron D, Rapoport TA. A membrane protein complex mediates retro-transloca- tion from the ER lumen into the cytosol. Nature. 2004; 429(6994):841–7.
• 23. Shen Y, Ballar P, Fang S. Ubiquitin ligase gp78 increases solubility and facilitates degradation of the Z variant of alpha-1-antitrypsin. Biochemical and biophysical research communications. 2006; 349 (4):1285–93.
• 24. Ye Y. Diverse functions with a common regulator: ubiquitin takes command of an AAA ATPase. Journal of structural biology. 2006; 156(1):29–40.
• 25. Zhong X, Shen Y, Ballar P, et al. AAA ATPase p97/valosin-containing protein interacts with gp78, a ubiquitin ligase for endoplasmic reticulum-associated degradation. The Journal of biological chemistry. 2004; 279(44):45676–84.
• 26. Song BL, Sever N, DeBose-Boyd RA. Gp78, a membrane-anchored ubiquitin ligase, associates with Insig-1 and couples sterol-regulated ubiquitination to degradation of HMG CoA reductase. Molecular cell. 2005; 19(6):829–40.
• 27. Ballar P, Shen Y, Yang H, Fang S. The role of a novel p97/valosin-containing protein-interacting motif of gp78 in endoplasmic reticulum-associated degradation. The Journal of biological chemistry. 2006; 281 (46):35359–68.
• 28. Ballar P, Zhong Y, Nagahama M, et al. Identification of SVIP as an endogenous inhibitor of endoplasmic reticulum-associated degradation. The Journal of biological chemistry. 2007; 282(47):33908–14.
• 29. Wu J, Peng D, Voehler M, Sanders CR, Li J. Structure and expression of a novel compact myelin pro- tein—small VCP-interacting protein (SVIP). Biochemical and biophysical research communications. 2013; 440(1):173–8.
• 30. Ballar P, Fang S. Regulation of ER-associated degradation via p97/VCP-interacting motif. Biochemical Society transactions. 2008; 36(Pt 5):818–22.
• 31. Wang Y, Ballar P, Zhong Y, et al. SVIP induces localization of p97/VCP to the plasma and lysosomal membranes and regulates autophagy. PloS one. 2011; 6(8):e24478.
• 32. Mizuno T, Suzuki N, Makino H, et al. Cancer stem-like cells of ovarian clear cell carcinoma are enriched in the ALDH-high population associated with an accelerated scavenging system in reactive oxygen species. Gynecol Oncol. 2015 May;137(2):299-305.
• 33. Xu C, Bailly-Maitre B and Reed JC. Endoplasmic reticulum stress: cell life and death decisions. The Journal of Clinical Investigation. 2005; 115(10):2656-2664.
• 34. Denmeade SR and Isaacs JT. The SERCA pump as a therapeutic target: making a “smart bomb” for prostate cancer. Cancer Biology & Therapy. 2005; 4(1):14-22.
• 35. Wang W.A, Groenendyk, J, Michalak M. Endoplasmic reticulum stress associated responses in cancer. Biochim. Biophys. Acta. 2014;1843, 2143–2149.
• 36. Salaroglio, I.C, Panada E, Moiso E, et al. PERK induces resistance to cell death elicited by endoplasmic reticulum stress and chemotherapy. Mol. Cancer 2017, 16, 91.
• 37. Bi M, Naczki C, Koritzinsky M, et al. ER stress-regulated translation increases tolerance to extreme hypoxia and promotes tumor growth. EMBO J. 2005, 24, 3470–3481.
• 38. Samanta S, Tamura S, Dubeau L, et al. Clinicopathological significance of endoplasmic reticulum stress proteins in ovarian carcinoma. Sci Rep. 2020 Feb 7;10(1):2160.
• 39. Ballar P, Shen Y, Yang H, Fang S. The role of a novel p97/valosin-containing protein-interacting motif of gp78 in endoplasmic reticulum-associated degradation. J Biol Chem. 2006 Nov 17;281(46):35359-68.
• 40. Nagahama M, Suzuki M, Hamada Y, et al. SVIP is a novel VCP/p97-interacting protein whose expression causes cell vacuolation. Mol Biol Cell. 2003 Jan;14(1):262-73.
• 41. Maurizi MR, Li CC. AAA proteins: in search of a common molecular basis. International Meeting on Cellular Functions of AAA Proteins. EMBO Rep. 2001 Nov;2(11):980-5.
• 42. Llinàs-Arias P, Rosselló-Tortella M, López-Serra P, et al. Epigenetic loss of the endoplasmic reticulum-associated degradation inhibitor SVIP induces cancer cell metabolic reprogramming. JCI Insight. 2019; 5 (8): e125888.
• 43. Bao D, Cheng C, Lan X, et al. Regulation of p53wt glioma cell proliferation by androgen receptor-mediated inhibition of small VCP/p97-interacting protein expression. Oncotarget. 2017; 8 (14): 23142-23154.
• 44. Erzurumlu Y, Ballar P. Androgen Mediated Regulation of Endoplasmic Reticulum-Associated Degradation and its Effects on Prostate Cancer. Scientific Reports 2017; 7: 40719.