Mitochondrial reactive oxygen species in TNFα-induced necroptosis Ji Young Lee, and Sang Won Kang Department of Life Science, Ewha Womans University, Seoul 03760, Korea BACKGROUND AIM Cell death is essential for homeostasis in organisms on removing damaged, degenerated or infected cells. Balance The role of Reactive Oxygen Species (ROS) is crucial in the regulation of various cellular between cell death and cell proliferation is important. Resistance to cell death can lead to cancer and other diseases, activity including cell death. ROS is a general term containing chemical species derived
while uncontrolled cell death can lead to a variety of diseases, including necrotic-related and autoimmune diseases. from oxygen. However, it is still unclear as to which specific type of ROS is involved in cell Later, as research into cell death mechanisms and signal transduction pathways progressed, cell death is classified into regulated cell death (RCD) and accidental cell death (ACD). ACD is triggered by unexpected damage and injury death pathway, leading to arguments arising about the classification of ROS in and it is biologically uncontrolled. In contrast, RCD has specific signaling
cascades, so RCD can be regulated by necroptosis. Here, we show that tumor necrosis factor-α (TNFα)-induced necroptosis defined effector molecules executing specific pathway. Reactive oxygen species (ROS) is generated from normal accompanies with mitochondrial superoxide anion. Similar to necroptosis, mitochondria cellular activity and plays an important role in various cellular activities, including cell differentiation and gene superoxide anion is regulated by a necroptosis kinase, receptor-interacting protein 3 expression. ROS includes the superoxide anion (O2˙ˉ), hydrogen peroxide (H2O2),
and hydroxyl radical (OH). ROS (RIP3). Since the level of mitochondrial superoxide anion increases in TNFα-induced is not only a product of cellular activity but also known as a second messenger that plays an important role in signal transduction such as specific cytokines and growth factors. Since previous study showed that the 2-Cys necroptosis, we investigated NADPH oxidase (NOX) as one of ROS generating systems peroxiredoxin (Prx) isoforms, Prx I and Prx II, regulate different apoptosis pathways via H2O2, I wonder which type of in cells. APX-115 (NOX inhibitor) decreases level of
mitochondrial superoxide anion ROS is involved in what process in necroptosis. Reactive oxygen species (ROS) is robustly produced during the cell generated in TNFα-induced necroptosis. More importantly, we showed that the inhibition death. However, few studies have identified types of ROS in different cell death pathways. In this study, I found of mitochondrial superoxide anion production does not contribute to necroptosis directly. difference of ROS type dependent on cell death pathway and their mechanism. Unexpectedly, mitochondrial Overall, this study implicates mitochondrial superoxide
anion in TNFα-induced necroptosis superoxide anion is produced by NOX in TNFα-induced necroptosis. Thus, this study demonstrates NOX is source of mitochondrial superoxide anion TNFα-induced necroptosis and it does not contribute to cell death directly. to be the product of TNFα-induced necroptosis. RESULTS CONCLUSION METHODS Mitochondrial superoxide occurred during TNFα-induced necroptosis in RIP3 Cell death assay NADH/NAD+ assay expression positive cells, and NOX was involved in the process. Although NOX Measurement of intracellular ROS Immunoblotting inhibitor reduced level of mitochondrial
superoxide anion, TNFα-induced necroptosis REFERENCES was not affected same as scavengers. On the other hand, when SOD2 was deleted and mitochondrial superoxide anion was increased, there was no effect on Grootjans, S., Vanden Berghe, T., and Vandenabeele, P. (2017). Initiation and execution mechanisms of necroptosis: an overview. Cell Death Differ necroptosis. Through these results suggested that mitochondrial superoxide anion 24, 1184-1195. does not contribute to necroptosis directly but it would be product of TNFα-induced Lee, S., Lee, J.Y., Lee, E.W., Park, S., Kang, D.H., Min, C., Lee,
D.J., Kang, necroptosis. The relationship between necroptosis and NOX has not been clearly D., Song, J., Kwon, J., et al. (2019). Absence of Cytosolic 2-Cys Prx clarified. Which NOX isoform plays a role in TNFα-induced necroptosis is not yet Subtypes I and II Exacerbates TNF-alpha-Induced Apoptosis via Different clear. Further studies are required to confirm interaction NOX and necroptosis. This Routes. Cell Rep 26, 2194-2211 e2196 finding will be a valuable discovery of a novel necroptosis regulatory mechanism. Contact information Here, we present evidence that increased mitochondrial
superoxide anion would be the product rather than the cause of TNFα-induced necroptosis. Ji Young Lee: lwldud96@ewhain.net [G. Cell differentiation, division, and death-4] Role of mitochondrial reactive oxygen species in TNF-α- induced necroptosis Jiyoung Lee¹, Sang Won Kang¹˙* ¹Department of Life Science, Ewha Womans University, seoul 03760, south korea Cell death is indispensable for maintaining homeostasis in organism. Because unregulated cell death causes various diseases such as cancer and autoimmune diseases, it is important to study the mechanisms to control cell death. Reactive oxygen
species (ROS) are robustly produced during the cell death. There are three types of ROS, such as superoxide anion (O2˙ˉ), hydrogen peroxide (H2O2), and hydroxyl radical (OH). However, few studies have identified types of ROS in different cell death pathways. Since we have shown that the 2-Cys peroxiredoxin (Prx) isoforms, Prx I and Prx II, regulate different apoptosis pathways via H2O2, a question was raised whether mitochondrial ROS is involved in the necroptosis. In this study, we show that different types of ROS are produced in the TNFα-induced apoptosis and necroptosis. Specifically, the
TNFα-induced necroptosis accompanies with mitochondrial superoxide anion. Moreover, the mitochondria superoxide anion was regulated by a necroptosis kinase RIPK3. Importantly, blocking mitochondrial superoxide production did not affect the necroptosis. Thus, the study implicates a selectivity on ROS production depending on cell death programs. Effect of berberine on the differentiation induction of acute promyelocytic leukemia (APL) cells 1 Hyein Noh , Aram Lee , Jihyun Lim , and Jong-Seok Lim 1,2 1 1 2 1 Department of Biological Science, Research Institute of Women’s Health and Cellular
Heterogeneity Research Center, Sookmyung Women’s University, Seoul 04310, Republic of Korea Abstract Figure 4 Berberine induced differentiation on RA- resistant HL-60 cells Acute promyelocytic leukemia (APL) is characterized by the (A) RA-resistant HL-60 cells were stained with anti-CD38 abnormal accumulation and rapid proliferation of atypical antibody. CD38 expression was analyzed by flow cytometry. promyelocytes that have impaired differentiation. (B) WT HL-60 and two RA-resistant cells (R38+ and R38-) Differentiation therapy with all-trans retinoic acid (ATRA) is were treated with 1 μM of
ATRA for 24 h. CD11b expression commonly applied to treat APL patients, but the long-term was analyzed using flow cytometry. (B) WT HL-60 and (C, D) therapy with ATRA easily induces drug resistance. Berberine two RA-resistant HL-60 cells were treated with 10 μM (BBR) is natural isoquinoline alkaloid that is known to have berberine and ATRA (0.5 and 1 μM) for 24-48 h. CD11b antitumor properties by affecting various biological functions. expression was analyzed using flow cytometry. In the present study, we demonstrate the anti-tumor effects of BBR in the human APL cell line HL-60. BBR in
combination with ATRA significantly increased protein levels of differentiation markers including PU.1. BBR also significantly increased the population of CD11b-positive cells and it showed a synergistic effect in combination with low concentration of ATRA. BBR increased phosphorylation of AMPK as HL-60 differentiation was in progress. When compound C, an AMPK inhibitor, was combined with BBR, the differentiation induced by BBR was significantly inhibited. BBR also induced differentiation of the ATRA-resistant HL- 60 cell line. In conclusion, these findings suggest that BBR- mediated
differentiation of HL-60 is affected by AMPK activation and BBR is expected to be a new therapeutic option for APL patients. Figure 2. AMPK inhibition attenuated the berberine- Purpose mediated differentiation of HL-60 cells (A) HL-60 cells were treated with berberine (10 μM) and Because of impaired hematopoietic differentiation, induction ATRA (0.5 μM) for 15-60 min. The protein level of pAMPK of differentiation of leukemic cells by differentiation therapy and GAPDH was measured by western blot. (B) HL-60 cells has been proven effective for APL patients. All-trans retinoic were treatedwith
AICAR(2.5and 5 mM) andATRA(0.25 acid (ATRA) is a commonly used differentiation-inducing and 0.5 μM) for 48 h. (A) CD11b expression was analyzed agent to treat APL patients. However, ATRA resistance using flow cytometry. (C) HL-60 cells were treated with commonly occurs in patients treated continuously with ATRA Compound C (0.5 and1μM) andATRA (0.5and 1μM) for as single agent. Combination of ATRA with anti-cancer 48 h. (A) CD11b expression were analyzed using flow chemotherapy can reduce the relapse rate while maintaining cytometry (D) HL-60 cells were treated with Compound C the complete
remission (CR) rate, resulting in improved (0.5 and 1 μM) and berberine (5 and 10 μM) for 48 h. CD11b Figure 5. CD38 expression in HL-60 cells was up- treatment performance. Berberine has various anti-cancer expression was analyzed using flow cytometry. ** p <0.01, regulated by berberine effects, including inhibition of proliferation and metastasis, *** p <0.001. (A) Wile type HL-60 was treated with 0.5 μM of ATRA and induction of apoptosis and differentiation. However, studies berberine (5 and 10 μM) for 48 h. CD38 expression was on the differentiation-inducing effects of berberine in AML
analyzed using flow cytometry. (B) R38- and (C) R38+, two are still insufficient. The main purpose of this study was to RA-resistant HL-60 cell lines, were treated with 1 μM of identify differentiation inducing effects of berberine in APL ATRA and berberine (5 and 10 μM) for 48 h. CD38 and reveal its molecular mechanisms. expression was analyzed using flow cytometry. Results Conclusion 1. Berberine increases expression of PU.1 and population of Figure 3. Effect of AMPK α1 overexpression on CD11b positive cell in HL-60 cells. differentiation of HL-60 cells HL-60 cells were transiently
transfected with the vector 2. Differentiation, which is induced by berberine, is affected control (mock) or AMPK α1 WT plasmid DNA. (A) The by AMPK activation indirectly. experimental schedule for transfection. HL-60 cells were transfected with plasma DNA for 18 h and treated with 3. Berberine increases expression of CD38, which is DMSO or ATRA (1 μM) for 24 h. (B, C) The protein levels involved in drug resistance, during differentiation. of AMPK, pAMPK and GAPDH were analyzed by western blot. (C, D) The protein levels of PU.1 were analyzed by western blot. CD11b expression was analyzed using
flow References cytometry. 1. Khwaja,A., Bjorkholm,M., Gale,R.E., Levine,R.L., Jordan,C.T., Ehninger,G., Bloomfield,C.D., Estey, E., Burnett, A., Cornelissen, J. J., Scheinberg, D. A., Bouscary, D., & Linch, D. C. 2016. "Acute myeloid leukaemia". Nature Reviews Disease Primers, 2(1), 16010. 2. Liu, D., Meng, X., Wu, D., Qiu, Z., & Luo, H. 2019. "A natural isoquinoline alkaloid with antitumor activity: Studies of the biological activities of berberine". Frontiers Figure 1. Berberine induced differentiation of HL-60 cells in Pharmacology, 10, 9. Cells were treated with berberine (2.5, 5, 10, and
20 µM), 3. Jacquel, A., Luciano, F., Robert, G., & Auberger, P.
