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Overexpression of SIRT6 alleviates apoptosis and enhances cell viability and monoclonal antibody expression in CHO-K1 cells

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Abstract

Background

Chinese hamster ovary (CHO) cells are the most predominantly utilized host for the production of monoclonal antibodies (mAbs) and other complex glycoproteins. A major challenge in the process of CHO cell culture is the occurrence of cell death following different stressful conditions, which hinders the production yield. Engineering genes involved in pathways related to cell death is a remarkable strategy to delay apoptosis, improve cell viability and enhance productivity. SIRT6 is a stress-responsive protein that regulates DNA repair, maintains genome integrity, and is critical for longevity and cell survival in organisms.

Methods and results

In this study, SIRT6 was stably overexpressed in CHO-K1 cells and the impact of its expression on apoptosis related gene expression profile, viability, apoptosis, and mAb productivity was investigated. While a significant increase was observed in Bcl-2 mRNA level, caspase-3 and Bax mRNA levels were decreased in the SIRT6 engineered cells compared to the parental CHO-K1 cells. Moreover, improved cell viability and decreased rate of apoptotic progression was observed in a SIRT6-derived clone in comparision to the CHO-K1 cells during 5 days of batch culture. anti-CD52 IgG1 mAb titers were improved up to 1.7- and 2.8-fold in SIRT6-derived clone during transient and stable expression, respectively.

Conclusions

This study indicates the positive effects of SIRT6 overexpression on cell viability and anti-CD52 IgG1 mAb expression in CHO-K1 cells. Further studies are needed to examine the potential of SIRT6-engineered host cells for the production of recombinant biotherapeutics in industrial settings.

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Data Availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Abbreviations

CHO:

Chinese hamster ovary

mAb:

monoclonal antibody

Bcl-2:

B-cell lymphoma 2

Bax:

BCL2-Associated X

BAK1:

BCL2 Antagonist/Killer 1

Bcl-xL:

B-cell lymphoma-extra large

PARP-1:

poly [ADP-ribose] polymerase 1

Gcn5:

general control non-derepressible 5

PGC-1α:

Peroxisome proliferator-activated receptor-gamma coactivator

qRT-PCR:

quantitative real‑time PCR

References

  1. Xiao S, Li Q, Jiang J, Huo C, Chen H and Guo M (2023) Rapid Identification of chinese Hamster ovary cell apoptosis and its potential role in process Robustness Assessment. Bioengineering (Basel) 10. https://doi.org/10.3390/bioengineering10030357

  2. Bryan L, Clynes M and Meleady P (2021) The emerging role of cellular post-translational modifications in modulating growth and productivity of recombinant chinese hamster ovary cells. Biotechnology Advances 49:107757. https://doi.org/10.1016/j.biotechadv.2021.107757

    Article  CAS  PubMed  Google Scholar 

  3. Dumont J, Euwart D, Mei B, Estes S and Kshirsagar R (2016) Human cell lines for biopharmaceutical manufacturing: history, status, and future perspectives. Critical reviews in biotechnology 36:1110–1122.

    Article  CAS  PubMed  Google Scholar 

  4. Tihanyi B and Nyitray L (2020) Recent advances in CHO cell line development for recombinant protein production. Drug Discovery Today: Technologies 38:25–34. https://doi.org/10.3109/07388551.2015.1084266

    Article  CAS  PubMed  Google Scholar 

  5. Chevallier V, Andersen MR and Malphettes L (2020) Oxidative stress-alleviating strategies to improve recombinant protein production in CHO cells. Biotechnology and bioengineering 117:1172–1186. https://doi.org/10.1002/bit.27247

    Article  CAS  PubMed  Google Scholar 

  6. Zhan C, Bidkhori G, Schwarz H, Malm M, Mebrahtu A, Field R, Sellick C, Hatton D, Varley P and Mardinoglu A (2020) Low shear stress increases recombinant protein production and high shear stress increases apoptosis in human cells. Iscience 23:101653. https://doi.org/10.1016/j.isci.2020.101653

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Kyeong M and Lee JS (2022) Endogenous BiP reporter system for simultaneous identification of ER stress and antibody production in chinese hamster ovary cells. Metab Eng 72:35–45. https://doi.org/10.1016/j.ymben.2022.02.002

    Article  CAS  PubMed  Google Scholar 

  8. Kuo C-C, Chiang AW, Shamie I, Samoudi M, Gutierrez JM and Lewis NE (2018) The emerging role of systems biology for engineering protein production in CHO cells. Current Opinion in Biotechnology 51:64–69. https://doi.org/10.1016/j.copbio.2017.11.015

    Article  CAS  PubMed  Google Scholar 

  9. Lee JS, Ha TK, Park JH and Lee GM (2013) Anti-cell death engineering of CHO cells: Co‐overexpression of Bcl‐2 for apoptosis inhibition, Beclin‐1 for autophagy induction. Biotechnology and bioengineering 110:2195–2207. https://doi.org/10.1002/bit.24879

    Article  CAS  PubMed  Google Scholar 

  10. Zhang X, Han L, Zong H, Ding K, Yuan Y, Bai J, Zhou Y, Zhang B and Zhu J (2018) Enhanced production of anti-PD1 antibody in CHO cells through transient co-transfection with anti-apoptotic genes Bcl-x L and Mcl-1. Bioprocess and biosystems engineering 41:633–640. https://doi.org/10.1007/s00449-018-1898-z

    Article  CAS  PubMed  Google Scholar 

  11. Tang D, Lam C, Bauer N, Auslaender S, Snedecor B, Laird MW and Misaghi S (2022) Bax and bak knockout apoptosis-resistant chinese hamster ovary cell lines significantly improve culture viability and titer in intensified fed‐batch culture process. Biotechnology Progress 38:e3228. https://doi.org/10.1002/btpr.3228

    Article  CAS  PubMed  Google Scholar 

  12. Xiong K, Marquart KF, la Cour Karottki KJ, Li S, Shamie I, Lee JS, Gerling S, Yeo NC, Chavez A and Lee GM (2019) Reduced apoptosis in chinese hamster ovary cells via optimized CRISPR interference. Biotechnology and bioengineering 116:1813–1819. https://doi.org/10.1002/bit.26969

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Yun CY, Liu S, Lim SF, Wang T, Chung BY, Teo JJ, Chuan KH, Soon AS, Goh KS and Song Z (2007) Specific inhibition of caspase-8 and-9 in CHO cells enhances cell viability in batch and fed-batch cultures. Metabolic engineering 9:406–418. https://doi.org/10.1016/j.ymben.2007.06.001

    Article  CAS  PubMed  Google Scholar 

  14. Kang SJ and Rhee WJ (2019) Silkworm Storage protein 1 inhibits autophagy-mediated apoptosis. Int J Mol Sci 20. https://doi.org/10.3390/ijms20020318

  15. Galluzzi L, Vitale I, Aaronson SA, Abrams JM, Adam D, Agostinis P, Alnemri ES, Altucci L, Amelio I and Andrews DW (2018) Molecular mechanisms of cell death: recommendations of the nomenclature Committee on Cell Death 2018. Cell Death & Differentiation 25:486–541. https://doi.org/10.1038/s41418-017-0012-4

    Article  Google Scholar 

  16. Fiorentino F, Mai A and Rotili D (2021) Emerging therapeutic potential of SIRT6 modulators. Journal of Medicinal Chemistry 64:9732–9758. https://doi.org/10.1021/acs.jmedchem.1c00601

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Baeken MW (2023) Sirtuins and their influence on autophagy. J Cell Biochem. https://doi.org/10.1002/jcb.30377

    Article  PubMed  Google Scholar 

  18. Tian X, Firsanov D, Zhang Z, Cheng Y, Luo L, Tombline G, Tan R, Simon M, Henderson S and Steffan J (2019) SIRT6 is responsible for more efficient DNA double-strand break repair in long-lived species. Cell 177:622–638. e22. https://doi.org/10.1016/j.cell.2019.03.043

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Wu Y, Chen L, Wang Y, Li W, Lin Y, Yu D, Zhang L, Li F and Pan Z (2015) Overexpression of Sirtuin 6 suppresses cellular senescence and NF-κB mediated inflammatory responses in osteoarthritis development. Scientific reports 5:1–11. https://doi.org/10.1038/srep17602

    Article  CAS  Google Scholar 

  20. Chang AR, Ferrer CM and Mostoslavsky R (2020) SIRT6, a mammalian deacylase with multitasking abilities. Physiological reviews 100:145–169. https://doi.org/10.1152/physrev.00030.2018

    Article  CAS  PubMed  Google Scholar 

  21. Peshti V, Obolensky A, Nahum L, Kanfi Y, Rathaus M, Avraham M, Tinman S, Alt FW, Banin E and Cohen HY (2017) Characterization of physiological defects in adult SIRT6-/-mice. PloS one 12:e0176371. https://doi.org/10.1371/journal.pone.0176371.t001

    Article  PubMed  PubMed Central  Google Scholar 

  22. Roichman A, Kanfi Y, Glazz R, Naiman S, Amit U, Landa N, Tinman S, Stein I, Pikarsky E and Leor J (2017) SIRT6 overexpression improves various aspects of mouse healthspan. The Journals of Gerontology: Series A 72:603–615. https://doi.org/10.1093/gerona/glw152

    Article  CAS  Google Scholar 

  23. Liu G, Chen H, Liu H, Zhang W and Zhou J (2021) Emerging roles of SIRT6 in human diseases and its modulators. Medicinal research reviews 41:1089–1137. https://doi.org/10.1002/med.21753

    Article  PubMed  Google Scholar 

  24. Xiong X, Sun X, Wang Q, Qian X, Zhang Y, Pan X and Dong XC (2016) SIRT6 protects against palmitate-induced pancreatic β-cell dysfunction and apoptosis. The Journal of endocrinology 231:159. https://doi.org/10.1530/JOE-16-0317

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Fiorentino F, Carafa V, Favale G, Altucci L, Mai A and Rotili D (2021) The two-faced role of SIRT6 in cancer. Cancers 13:1156. https://doi.org/10.3390/cancers13051156

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Chen J, Xie J-J, Jin M-Y, Gu Y-T, Wu C-C, Guo W-J, Yan Y-Z, Zhang Z-J, Wang J-L and Zhang X-L (2018) Sirt6 overexpression suppresses senescence and apoptosis of nucleus pulposus cells by inducing autophagy in a model of intervertebral disc degeneration. Cell death & disease 9:56. https://doi.org/10.1038/s41419-017-0085-5

    Article  CAS  Google Scholar 

  27. Naderi F, Hashemi M, Bayat H, Mohammadian O, Pourmaleki Eh, Etemadzadeh MH and Rahimpour A (2018) The augmenting effects of the tDNA insulator on stable expression of monoclonal antibody in chinese hamster ovary cells. Monoclonal Antibodies in Immunodiagnosis and Immunotherapy 37:200–206. https://doi.org/10.1089/mab.2018.0015

    Article  CAS  PubMed  Google Scholar 

  28. Mohammadian O, Rajabibazl M, Pourmaleki E, Bayat H, Ahani R and Rahimpour A (2019) Development of an improved lentiviral based vector system for the stable expression of monoclonal antibody in CHO cells. Prep Biochem Biotechnol 49:822–829. https://doi.org/10.1080/10826068.2019.1621893

    Article  CAS  PubMed  Google Scholar 

  29. Henry MN, MacDonald MA, Orellana CA, Gray PP, Gillard M, Baker K, Nielsen LK, Marcellin E, Mahler S and Martínez VS (2020) Attenuating apoptosis in chinese hamster ovary cells for improved biopharmaceutical production. Biotechnology and Bioengineering 117:1187–1203. https://doi.org/10.1002/bit.27269

    Article  CAS  PubMed  Google Scholar 

  30. Weinguny M, Eisenhut P, Klanert G, Virgolini N, Marx N, Jonsson A, Ivansson D, Lövgren A and Borth N (2020) Random epigenetic modulation of CHO cells by repeated knockdown of DNA methyltransferases increases population diversity and enables sorting of cells with higher production capacities. Biotechnology and bioengineering 117:3435–3447. https://doi.org/10.1002/bit.27493

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Taylor JR, Wood JG, Mizerak E, Hinthorn S, Liu J, Finn M, Gordon S, Zingas L, Chang C and Klein MA (2022) Sirt6 regulates lifespan in Drosophila melanogaster. Proceedings of the National Academy of Sciences 119:e2111176119. https://doi.org/10.1073/pnas.2111176119

  32. Spahn PN, Zhang X, Hu Q, Lu H, Hamaker NK, Hefzi H, Li S, Kuo CC, Huang Y and Lee JC (2022) Restoration of DNA repair mitigates genome instability and increases productivity of chinese hamster ovary cells. Biotechnology and Bioengineering 119:963–982. https://doi.org/10.1002/bit.28016

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Lee JC (2020) Investigating the use of DNA repair as a strategy to enhance production stability in Chinese.

  34. Zhang C, Yu Y, Huang Q and Tang K (2019) SIRT6 regulates the proliferation and apoptosis of hepatocellular carcinoma via the ERK1/2 signaling pathway. Molecular Medicine Reports 20:1575–1582. https://doi.org/10.3892/mmr.2019.10398

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Ran L-K, Chen Y, Zhang Z-Z, Tao N-N, Ren J-H, Zhou L, Tang H, Chen X, Chen K and Li W-Y (2016) SIRT6 overexpression potentiates apoptosis evasion in Hepatocellular Carcinoma via BCL2-Associated X protein–dependent apoptotic PathwayThe role of SIRT6 in HCC. Clinical Cancer Research 22:3372–3382. https://doi.org/10.1158/1078-0432.CCR-15-1638

    Article  CAS  PubMed  Google Scholar 

  36. Fan Y, Cheng J, Yang Q, Feng J, Hu J, Ren Z, Yang H, Yang D and Ding G (2021) Sirt6-mediated Nrf2/HO-1 activation alleviates angiotensin II-induced DNA DSBs and apoptosis in podocytes. Food & Function 12:7867–7882. https://doi.org/10.1039/D0FO03467C

    Article  CAS  Google Scholar 

  37. Fan Y, Yang Q, Yang Y, Gao Z, Ma Y, Zhang L, Liang W and Ding G (2019) Sirt6 suppresses high glucose-induced mitochondrial dysfunction and apoptosis in podocytes through AMPK activation. International journal of biological sciences 15:701. https://doi.org/10.7150/ijbs.29323

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Li Z-M, Fan Z-L, Wang X-Y and Wang T-Y (2022) Factors affecting the expression of recombinant protein and improvement strategies in chinese Hamster ovary cells. Frontiers in Bioengineering and Biotechnology 10. https://doi.org/10.3389/fbioe.2022.880155

  39. Li Y, Zhang X, Wang L, Zong H, Yuan Y, Han L, Li X, Xu C, Zhang J, Zhu J and Zhang B (2019) Enhanced production of Anti-PD1 antibody in CHO cells through transient co-transfection with anti-apoptotic gene Bcl-xL combined with rapamycin. Processes 7:9. https://doi.org/10.1007/s00449-018-1898-z

    Article  CAS  Google Scholar 

  40. Lu Y, Zhou Q, Han Q, Wu P, Zhang L, Zhu L, Weaver DT, Xu C and Zhang B (2018) Inactivation of deubiquitinase CYLD enhances therapeutic antibody production in chinese hamster ovary cells. Appl Microbiol Biotechnol 102:6081–6093. https://doi.org/10.1007/s00253-018-9070-x

    Article  CAS  PubMed  Google Scholar 

  41. Xu WJ, Lin Y, Mi CL, Pang JY and Wang TY (2023) Progress in fed-batch culture for recombinant protein production in CHO cells. Appl Microbiol Biotechnol 107:1063–1075. https://doi.org/10.1007/s00253-022-12342-x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Xin Y, Xu L, Zhang X, Yang C, Wang Q and Xiong X (2021) Sirtuin 6 ameliorates alcohol-induced liver injury by reducing endoplasmic reticulum stress in mice. Biochem Biophys Res Commun 544:44–51. https://doi.org/10.1016/j.bbrc.2021.01.061

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

Authors wish to thank School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences for their support.

Funding

This study was supported by School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran (grants No. 15570 and 166).

Author information

Authors and Affiliations

  1. Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran

    Nader Hashemi, Forough Shams, Bahram Kazemi & Javad Ranjbari

  2. Department of Life Science Engineering, Faculty of New Sciences and Technology, University of Tehran, Tehran, Iran

    Sayed Hassan Tabatabaee

  3. Medical Nanotechnology and Tissue Engineering Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran

    Azam Rahimpour

  4. Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran

    Azam Rahimpour

  5. Department of Clinical Biochemistry, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran

    Masoumeh Rajabibazl

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  1. Nader Hashemi
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Contributions

Experimental studies, data collection and data analysis were performed by NH. SHT and FS collaborated in experimental studies. Study conception, design, and supervision were performed by BK and AR. MR and JR contributed in data analysis and interpretation. The first draft of the manuscript was written by NH, and all authors commented on the previous drafts of the manuscript. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Azam Rahimpour or Bahram Kazemi.

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Hashemi, N., Tabatabaee, S.H., Shams, F. et al. Overexpression of SIRT6 alleviates apoptosis and enhances cell viability and monoclonal antibody expression in CHO-K1 cells. Mol Biol Rep 50, 6019–6027 (2023). https://doi.org/10.1007/s11033-023-08483-5

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