Histone H3 is a nucleosome scaffold protein that is involved in a variety of intracellular processes. Aberrant modification of H3 is important in carcinogenesis. In contrast, free histones in cells can act as stimuli to trigger cellular immune responses and cell death. In this study, we linked cell-penetrating peptide HIV Tat to a histone H3 fragment to achieve intracellular delivery in tumor cells. We found that Tat-conjugated histone polypeptides localized to nuclei of lung and breast cancer cells and caused cell death. A trans-configured Tat sequence displayed dramatically improved peptide half-life and cytotoxicity. Mechanistic studies demonstrated that treatment with the peptides significantly elevated mitogen-activated protein kinase (MAPK) signaling, reactive oxygen species (ROS) production, as well as levels of stress-inducible transcription factor ATF3 (activating transcription factor 3) and AP-1 (activating protein-1). Cytotoxicity of the peptide was significantly reduced by inhibition of AP-1 activity and ROS production. These results suggest the potential of Tat-conjugated H3 peptides as antitumor agents to induce cell death via increased cellular stress response by activating p38-MAPK signaling and intracellular ROS production.
HeoK, KimJS, KimK, et al.Cell-penetrating H4 tail peptides potentiate p53-mediated transactivation via inhibition of G9a and HDAC1. Oncogene, 2013; 32(20):2510–2520; doi: 10.1038/onc.2012.273
4.
YuJR, LeRoyG, BreadyD, et al.The H3K36me2 writer-reader dependency in H3K27M-DIPG. Sci Adv, 2021;7(29):eabg7444; doi:10.1126/sciadv.abg7444
5.
AllamR, KumarSV, DarisipudiMN, et al.Extracellular histones in tissue injury and inflammation. J Mol Med (Berl), 2014; 92(5):465–472; doi: 10.1007/s00109-014-1148-z
6.
XuJ, ZhangX, PelayoR, et al.Extracellular histones are major mediators of death in sepsis. Nat Med, 2009; 15(11):1318–1321; doi: 10.1038/nm.2053
7.
Silvestre-RoigC, BrasterQ, WichapongK, et al.Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death. Nature, 2019; 569(7755):236–240; doi: 10.1038/s41586-019-1167-6
8.
BecharaC, SaganS. Cell-penetrating peptides: 20 Years later, where do we stand?. FEBS Lett, 2013; 587(12):1693–1702; doi: 10.1016/j.febslet.2013.04.031
9.
GreenM, LoewensteinPM. Autonomous functional domains of chemically synthesized human immunodeficiency virus tat trans-activator protein. Cell, 1988; 55(6):1179–1188.
10.
ParkJ, RyuJ, KimKA, et al.Mutational analysis of a human immunodeficiency virus type 1 Tat protein transduction domain which is required for delivery of an exogenous protein into mammalian cells. J Gen Virol, 2002; 83(Pt 5):1173–1181; doi: 10.1099/0022-1317-83-5-1173
11.
SchorderetDF, ManziV, CanolaK, et al.D-TAT transporter as an ocular peptide delivery system. Clin Exp Ophthalmol, 2005; 33(6):628–635; doi: 10.1111/j.1442-9071.2005.01108.x
HaiT, HartmanMG. The molecular biology and nomenclature of the activating transcription factor/cAMP responsive element binding family of transcription factors: Activating transcription factor proteins and homeostasis. Gene, 2001; 273(1):1–11; doi: 10.1016/s0378-1119(01)00551-0
14.
HessJ, AngelP, Schorpp-KistnerM. AP-1 subunits: Quarrel and harmony among siblings. J Cell Sci, 2004; 117(Pt 25):5965–5973; doi: 10.1242/jcs.01589
15.
HaiT, WolfgangCD, MarseeDK, et al.ATF3 and stress responses. Gene Expr, 1999; 7(4–6):321–335.
16.
SyedV, MukherjeeK, Lyons-WeilerJ, et al.Identification of ATF-3, caveolin-1, DLC-1, and NM23-H2 as putative antitumorigenic, progesterone-regulated genes for ovarian cancer cells by gene profiling. Oncogene, 2005; 24(10):1774–1787; doi: 10.1038/sj.onc.1207991
17.
MashimaT, UdagawaS, TsuruoT. Involvement of transcriptional repressor ATF3 in acceleration of caspase protease activation during DNA damaging agent-induced apoptosis. J Cell Physiol, 2001; 188(3):352–358; doi: 10.1002/jcp.1130
18.
EdagawaM, KawauchiJ, HirataM, et al.Role of activating transcription factor 3 (ATF3) in endoplasmic reticulum (ER) stress-induced sensitization of p53-deficient human colon cancer cells to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis through up-regulation of death receptor 5 (DR5) by zerumbone and celecoxib. J Biol Chem, 2014; 289(31):21544–21561; doi: 10.1074/jbc.M114.558890
19.
ShaulianE, KarinM. AP-1 as a regulator of cell life and death. Nat Cell Biol, 2002; 4(5):E131–E136; doi: 10.1038/ncb0502-e131
20.
ShaulianE, KarinM. AP-1 in cell proliferation and survival. Oncogene, 2001; 20(19):2390–2400; doi: 10.1038/sj.onc.1204383
21.
BrandtB, Abou-EladabEF, TiedgeM, et al.Role of the JNK/c-Jun/AP-1 signaling pathway in galectin-1-induced T-cell death. Cell Death Dis, 2010;1:e23; doi: 10.1038/cddis.2010.1
22.
LauricellaM, EmanueleS, D'AnneoA, et al.JNK and AP-1 mediate apoptosis induced by bortezomib in HepG2 cells via FasL/caspase-8 and mitochondria-dependent pathways. Apoptosis, 2006; 11(4):607–625; doi: 10.1007/s10495-006-4689-y
23.
CastellJV, Gómez-LechónMJ. In Vitro Methods in Pharmaceutical Research. Academic Press; 1996; pp. 129–153.
24.
TimmermannS, LehrmannH, PolesskayaA, et al.Histone acetylation and disease. Cell Mol Life Sci, 2001; 58(5–6):728–736; doi: 10.1007/pl00000896
25.
SvenssonEC, HugginsGS, DardikFB, et al.A functionally conserved N-terminal domain of the friend of GATA-2 (FOG-2) protein represses GATA4-dependent transcription. J Biol Chem, 2000; 275(27):20762–20769; doi: 10.1074/jbc.M001522200
26.
ZhaoX, QianL, QiD, et al.The 57th amino acid conveys the differential subcellular localization of human immunodeficiency virus-1 Tat derived from subtype B and C. Virus Genes, 2016; 52(2):179–188; doi: 10.1007/s11262-015-1267-9
27.
EferlR, WagnerEF. AP-1: A double-edged sword in tumorigenesis. Nat Rev Cancer, 2003; 3(11):859–868; doi: 10.1038/nrc1209
28.
BorgoniS, SofyaliE, SoleimaniM, et al.Time-resolved profiling reveals ATF3 as a novel mediator of endocrine resistance in breast cancer. Cancers (Basel), 2020;12(10):2918; doi: 10.3390/cancers12102918
29.
JohnsonGL, LapadatR. Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science, 2002; 298(5600):1911–1912; doi: 10.1126/science.1072682
30.
CagnolS, ChambardJC. ERK and cell death: Mechanisms of ERK-induced cell death—Apoptosis, autophagy and senescence. FEBS J, 2010; 277(1):2–21; doi: 10.1111/j.1742-4658.2009.07366.x
31.
Ray ChaudhuriA, NussenzweigA. The multifaceted roles of PARP1 in DNA repair and chromatin remodelling. Nat Rev Mol Cell Biol, 2017; 18(10):610–621; doi: 10.1038/nrm.2017.53
32.
KoHM, LeeSH, JeeW, et al.Gancaonin N from Glycyrrhiza uralensis attenuates the inflammatory response by downregulating the NF-kappaB/MAPK pathway on an acute pneumonia in vitro model. Pharmaceutics, 2021;13(7):1028; doi: 10.3390/pharmaceutics13071028
33.
SunKH, de PabloY, VincentF, et al.Novel genetic tools reveal Cdk5's major role in Golgi fragmentation in Alzheimer's disease. Mol Biol Cell, 2008; 19(7):3052–3069; doi: 10.1091/mbc.E07-11-1106
34.
BockFJ, TaitSWG. Mitochondria as multifaceted regulators of cell death. Nat Rev Mol Cell Biol, 2020; 21(2):85–100; doi: 10.1038/s41580-019-0173-8
35.
FinkelT. Signal transduction by reactive oxygen species. J Cell Biol, 2011; 194(1):7–15; doi: 10.1083/jcb.201102095
36.
Di CerboV, SchneiderR. Cancers with wrong HATs: The impact of acetylation. Brief Funct Genomics, 2013; 12(3):231–243; doi: 10.1093/bfgp/els065
37.
BrownR, StrathdeeG. Epigenomics and epigenetic therapy of cancer. Trends Mol Med, 2002; 8(4Suppl):S43–S48; doi: 10.1016/s1471-4914(02)02314-6
SankaranSM, WilkinsonAW, EliasJE, et al.A PWWP domain of histone-lysine N-methyltransferase NSD2 binds to dimethylated Lys-36 of histone H3 and regulates NSD2 function at chromatin. J Biol Chem, 2016; 291(16):8465–8474; doi: 10.1074/jbc.M116.720748
40.
ChiangCM.Brd4 engagement from chromatin targeting to transcriptional regulation: Selective contact with acetylated histone H3 and H4. F1000 Biol Rep, 2009;1:98; doi: 10.3410/B1-98
41.
BrooksH, LebleuB, VivesE. Tat peptide-mediated cellular delivery: Back to basics. Adv Drug Deliv Rev, 2005; 57(4):559–577; doi: 10.1016/j.addr.2004.12.001
Hossain-IbrahimMK, RezajooiK, MacNallyJK, et al.Effects of lipopolysaccharide-induced inflammation on expression of growth-associated genes by corticospinal neurons. BMC Neurosci, 2006;7:8; doi: 10.1186/1471-2202-7-8
44.
ZhaoJ, LiX, GuoM, et al.The common stress responsive transcription factor ATF3 binds genomic sites enriched with p300 and H3K27ac for transcriptional regulation. BMC Genomics 2016;17:335; doi: 10.1186/s12864-016-2664-8
45.
HaiT. The ATF Transcription Factors in Cellular Adaptive Responses. In: Ma, J. (eds) Gene Expression and Regulation. Springer: NY, 2006.
46.
ThompsonMR, XuD, WilliamsBR. ATF3 transcription factor and its emerging roles in immunity and cancer. J Mol Med (Berl), 2009; 87(11):1053–1060; doi: 10.1007/s00109-009-0520-x
47.
WoodgettJR, PulvererBJ, NikolakakiE, et al.Regulation of jun/AP-1 oncoproteins by protein phosphorylation. Adv Second Messenger Phosphoprotein Res, 1993; 28:261–269.
48.
HoetzeneckerW, EchtenacherB, GuenovaE, et al.ROS-induced ATF3 causes susceptibility to secondary infections during sepsis-associated immunosuppression. Nat Med, 2011; 18(1):128–134; doi: 10.1038/nm.2557
49.
Aharoni-SimonM, ReifenR, TiroshO. ROS-production-mediated activation of AP-1 but not NFkappaB inhibits glutamate-induced HT4 neuronal cell death. Antioxid Redox Signal, 2006; 8(7–8):1339–1349; doi: 10.1089/ars.2006.8.1339
50.
MonksTJ, XieR, TikooK, et al.Ros-induced histone modifications and their role in cell survival and cell death. Drug Metab Rev, 2006; 38(4):755–767; doi: 10.1080/03602530600959649
51.
MaoX, YuCR, LiWH, et al.Induction of apoptosis by shikonin through a ROS/JNK-mediated process in Bcr/Abl-positive chronic myelogenous leukemia (CML) cells. Cell Res, 2008; 18(8):879–888; doi: 10.1038/cr.2008.86
52.
TietzO, Cortezon-TamaritF, ChalkR, et al.Tricyclic cell-penetrating peptides for efficient delivery of functional antibodies into cancer cells. Nat Chem, 2022; 14(3):284–293; doi: 10.1038/s41557-021-00866-0
53.
ChengQ, WeiT, FarbiakL, et al.Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR-Cas gene editing. Nat Nanotechnol, 2020; 15(4):313–320; doi: 10.1038/s41565-020-0669-6
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