bims-polyam 2023-03-26 papers

Issue of 2023–03–26
six papers selected by
Sebastian J. Hofer, University of Graz

Appl Microbiol Biotechnol. 2023 Mar 22.

Enhanced cell growth, production, and mAb quality produced in Chinese hamster ovary-K1 cells by supplementing polyamine in the media.

Da Eun Kang, Yeong Bin An, Yeunju Kim, Seawon Ahn, Young Jin Kim, Jung Soo Lim, Soo Hyun Ryu, Hyoju Choi, Jiseon Yoo, Weon-Kyoo You, Dong-Yup Lee, Junsoo Park, Minsun Hong, Gyun Min Lee, Jong Youn Baik, Jong Kwang Hong.

Polyamines such as putrescine (PUT), spermidine (SPD), and spermine (SPM) are amine group-containing biomolecules that regulate multiple intracellular functions such as proliferation, differentiation, and stress response in mammalian cells. Although these biomolecules can be generated intracellularly, lack of polyamine-synthesizing activity has occasionally been reported in a few mammalian cell lines such as Chinese hamster ovary (CHO)-K1; thus, polyamine supplementation in serum-free media is required to support cell growth and production. In the present study, the effects of biogenic polyamines PUT, SPD, and SPM in media on cell growth, production, metabolism, and antibody quality were explored in cultures of antibody-producing CHO-K1 cells. Polyamine withdrawal from media significantly suppressed cell growth and production. On the other hand, enhanced culture performance was achieved in polyamine-containing media conditions in a dose-dependent manner regardless of polyamine type. In addition, in polyamine-deprived medium, distinguishing metabolic features, such as enriched glycolysis and suppressed amino acid consumption, were observed and accompanied by higher heterogeneity of antibody quality compared with the optimal concentration of polyamines. Furthermore, an excessive concentration of polyamines negatively affected culture performance as well as antibody quality. Hence, the results suggest that polyamine-related metabolism needs to be further investigated and polyamines in cell growth media should be optimized as a controllable parameter in CHO cell culture bioprocessing. KEY POINTS: • Polyamine supplementation enhanced cell growth and production in a dose-dependent manner • Polyamine type and concentration in the media affected mAb quality • Optimizing polyamines in the media is suggested in CHO cell bioprocessing.

Keywords: Antibody quality; CHO-K1 cells; Metabolism; Polyamine; Serum free media

DOI: https://doi.org/10.1007/s00253-023-12459-7

NMR Biomed. 2023 Mar 20. e4931

13 C NMR Quantification of Polyamine Syntheses in Rat Prostate.

Lindsey A Vandergrift, Nanbu Wang, Miry Zhu, Bailing Li, Shuyi Chen, Piet Habbel, Johannes Nowak, Ralph Mason, Aaron Grant, Yi Wang, Craig Malloy, Leo L Cheng.

BACKGROUND: Currently, many prostate cancer patients, detected through the prostatic specific antigen test, harbor organ-confined indolent disease that cannot be differentiated from aggressive cancer according to clinically and pathologically known measures. Spermine has been considered as an endogenous inhibitor for prostate-confined cancer growth and its expression has shown correlation with prostate cancer growth rates. If established clinically, measurements of spermine bio-synthesis rates in prostates may predict prostate cancer growth and patient outcomes. Using rat models, we tested the feasibility of quantifying spermine bio-synthesis rates with 13 C NMR.
METHODS: Male Copenhagen rats (10 weeks, n=6) were injected with uniformly 13 C-labeled L-ornithine HCl, and sacrificed in pairs at 10, 30 and 60 min after injection. Another two rats were injected with saline and sacrificed at 30 mins as controls. Prostates were harvested and extracted with perchloric acid and the neutralized solutions examined by 13 C NMR at 600 MHz.
RESULTS: 13 C NMR revealed measurable ornithine, as well as putrescine-spermidine-spermine syntheses in rat prostates allowing polyamine bio-synthetic and ornithine bio-catabolic rates to be calculated.
CONCLUSIONS: Our study demonstrated the feasibility of 13 C NMR in measuring bio-synthesis rates of ornithine to spermine enzymatic reactions in rat prostates. The current study established a foundation on which future investigations of protocols that differentiate prostate cancer growth rates according to the measure of ornithine to spermine bio-synthetic rates may be developed.

DOI: https://doi.org/10.1002/nbm.4931

Biomol Biomed. 2023 Mar 18.

Exogenous spermidine alleviates diabetic cardiomyopathy via suppressing ROS, ERS and Pannexin-1-mediated ferroptosis.

Jian Sun, Jiyu Xu, Yong Liu, Yitong Lin, Fengge Wang, Yue Han, Shumin Zhang, Xiaoyan Gao, Changqing Xu, Hui Yuan.

Diabetic cardiomyopathy (DCM) is a serious complication and death cause of diabetes mellitus (DM). Recent cardiology studies suggest that spermidine has cardioprotective effects. Here, we verified the hypothesis of spermidine's protective effects on DCM. Therefore, db/db mice and primary neonatal mouse cardiomyocytes were used to observe the effects of spermidine. Immunoblotting showed that ornithine decarboxylase (ODC) and SPD/spermine N1-acetyltransferase (SSAT) were downregulated and upregulated in the myocardium of db/db mice, respectively. We found that diabetic mice showed cardiac dysfunction in 12 weeks. Conversely, exogenous spermidine could improve cardiac functions and reduce the deposition of collagens, myocardial damage, reactive oxygen species (ROS) levels and endoplasmic reticulum stress (ERS) in diabetic mouse hearts. Our results also demonstrated that cardiomyocytes displayed ferroptosis and then activated Pannexin-1 expression, which resulted in the increase of the extracellular adenosine triphosphate (ATP). Subsequently, increased ATP as a paracrine molecule combined to purinergic receptor P2X7 (P2X7) to activate ERK1/2 signaling pathway in cardiomyocytes and activated NCOA4-mediated ferroptinophagy to promote lipid peroxidation and ferroptosis. Interestingly, spermidine could reverse these molecular processes. Our findings indicate an important new mechanism for DCM, and suggest spermidine have potential applicability to protect against deterioration of cardiac function with DCM.

DOI: https://doi.org/10.17305/bb.2022.8846

Iran J Pharm Res. 2022 Dec;21(1): e133776

Spermidine Alleviates Intrauterine Hypoxia-Induced Offspring Newborn Myocardial Mitochondrial Damage in Rats by Inhibiting Oxidative Stress and Regulating Mitochondrial Quality Control.

Nannan Chai, Haihong Zheng, Hao Zhang, Lingxu Li, Xue Yu, Liyi Wang, Xin Bi, Lihong Yang, Tongxu Niu, Xiujuan Liu, Yajun Zhao, Lijie Dong.

Background: Intrauterine hypoxia (IUH) increases the risk of cardiovascular diseases in offspring. As a reactive oxygen species (ROS) scavenger, polyamine spermidine (SPD) is essential for embryonic and fetal survival and growth. However, further studies on the SPD protection and mechanisms for IUH-induced heart damage in offspring are required.
Objectives: This study aimed to investigate the preventive effects of prenatal SPD treatment on IUH-induced heart damage in newborn offspring rats and its underlying mitochondrial-related mechanism.
Methods: The rat model of IUH was established by exposure to 10% O2 seven days before term. Meanwhile, for seven days, the pregnant rats were given SPD (5 mg.kg-1.d-1; ip). The one-day offspring rats were sacrificed to assess several parameters, including growth development, heart damage, cardiomyocytes proliferation, myocardial oxidative stress, cell apoptosis, and mitochondrial function, and have mitochondrial quality control (MQC), including mitophagy, mitochondrial biogenesis, and mitochondrial fusion/fission. In in vitro experiments, primary cardiomyocytes were subjected to hypoxia with or without SPD for 24 hours.
Results: IUH decreased body weight, heart weight, cardiac Ki67 expression, the activity of SOD, and the CAT and adenosine 5'-triphosphate (ATP) levels and increased the BAX/BCL2 expression, and TUNEL-positive nuclei numbers. Furthermore, IUH also caused mitochondrial structure abnormality, dysfunction, and decreased mitophagy (decreased number of mitophagosomes), declined mitochondrial biogenesis (decreased expression of SIRT-1, PGC-1α, NRF-2, and TFAM), and led to fission/fusion imbalance (increased percentage of mitochondrial fragments, increased DRP1 expression, and decreased MFN2 expression) in the myocardium. Surprisingly, SPD treatment normalized the variations in the IUH-induced parameters. Furthermore, SPD also prevented hypoxia-induced ROS accumulation, mitochondrial membrane potential decay, and the mitophagy decrease in cardiomyocytes.
Conclusion: Maternal SPD treatment caused IUH-induced heart damage in newborn offspring rats by improving the myocardial mitochondrial function via anti-oxidation and anti-apoptosis, and regulating MQC.

Keywords: Hypoxia; Mitochondrion; Myocardium; Oxidative Stress; Rats; Spermidine

DOI: https://doi.org/10.5812/ijpr-133776

EMBO Rep. 2023 Mar 21. e55373

pH regulates hematopoietic stem cell potential via polyamines.

Sachin Kumar, Jeffrey D Vassallo, Kalpana J Nattamai, Aishlin Hassan, Rebekah Karns, Angelika Vollmer, Karin Soller, Vadim Sakk, Mehmet Sacma, Travis Nemkov, Angelo D'Alessandro, Hartmut Geiger.

Upon ex vivo culture, hematopoietic stem cells (HSCs) quickly lose potential and differentiate into progenitors. The identification of culture conditions that maintain the potential of HSCs ex vivo is therefore of high clinical interest. Here, we demonstrate that the potential of murine and human HSCs is maintained when cultivated for 2 days ex vivo at a pH of 6.9, in contrast to cultivation at the commonly used pH of 7.4. When cultivated at a pH of 6.9, HSCs remain smaller, less metabolically active, less proliferative and show enhanced reconstitution ability upon transplantation compared to HSC cultivated at pH 7.4. HSCs kept at pH 6.9 show an attenuated polyamine pathway. Pharmacological inhibition of the polyamine pathway in HSCs cultivated at pH 7.4 with DFMO mimics phenotypes and potential of HSCs cultivated at pH 6.9. Ex vivo exposure to a pH of 6.9 is therefore a positive regulator of HSC function by reducing polyamines. These findings might improve HSC short-term cultivation protocols for transplantation and gene therapy interventions.

Keywords: DFMO; HSCs; ex vivo; pH; polyamine

DOI: https://doi.org/10.15252/embr.202255373

Tree Physiol. 2023 Mar 21. pii: tpad036. [Epub ahead of print]

The chances in the redox priming of nondormant recalcitrant seeds by spermidine.

Hanna Fuchs, Beata P Plitta-Michalak, Arleta Małecka, Liliana Ciszewska, Łukasz Sikorski, Aleksandra M Staszak, Marcin Michalak, Ewelina Ratajczak.

The problems posed by seed sensitivity to desiccation and aging have motivated the development of various techniques for mitigating their detrimental effects. The redox priming of seeds in antioxidant solution to improve their postharvest performance is one of the approaches. Spermidine (Spd) was tested as an invigorating solution on nondormant recalcitrant (desiccation sensitive) seeds of the silver maple (Acer saccharinum L.). The treatment resulted in an 8-10% increase in germination capacity in seeds subjected to mild and severe desiccation, while in aged seeds stored for six months, no significant change was observed. The cellular redox milieu, genetic stability, mitochondrial structure and function were investigated to provide information about the cellular targets of Spd activity. Spd improved the antioxidative capacity, especially the activity of catalase, and cellular membrane stability, protected genome integrity from oxidative damage and increased the efficiency of mitochondria. However, it also elicited a hydrogen peroxide burst. Therefore, it seems that redox priming in nondormant seeds that are highly sensitive to desiccation, although positively affected desiccated seed performance, may not be a simple solution to reinvigorate stored seeds with a low-efficiency antioxidant system.

Keywords: DNA damage; antioxidant system; reactive oxygen species; seed aging; seed desiccation; silver maple; spermidine

DOI: https://doi.org/10.1093/treephys/tpad036