








































International Journal of Cancer Therapy and Oncology
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Corresponding author: Keith R Davis; Biotechnology Program, Indiana University, Bloomington, Indiana, USA.
Cite this article as: Davis KR, Inaba J. Lunasin—a multifunctional anticancer peptide from soybean. Int J Cancer Ther Oncol. 2016;
4(2):4218. DOI: 10.14319/ijcto.42.18

© Davis et al. ISSN 2330-4049

Lunasin—a multifunctional anticancer peptide from soybean

Keith R. Davis1, Jun-ichi Inaba2

1Biotechnology Program, Indiana University, Bloomington, Indiana, USA
2Owensboro Cancer Research, University of Louisville, Louisville, Kentucky, USAReceived April 03, 2016; Revised June 15, 2016; Accepted June 20, 2016; Published Online June 30, 2016

Scientific Note
AbstractLunasin is a bioactive peptide that was originally isolated from soybean and hassince been shown to have a number of biological activities, including both cancerchemopreventive and therapeutic activities. Our recent focus has been ondetermining the range of cancer types that lunasin can affect and the mechanism ofaction against specific cancers. We recently found that lunasin has significanttherapeutic activity against non-small cell lung cancer (NSCLC) both in vitro and invivo. Mechanistic studies using lunasin-sensitive and lunasin-resistant NSCLC celllines revealed the lunasin blocks cell proliferation by inhibiting cell cycleprogression at the G1/S phase interface and that this inhibition was associatedwith reduced Akt signaling. In addition, we found that these effects were linked tothe inhibition of integrin signaling through αv-containing integrins. Our resultsprovide strong support for the hypothesis that direct effects on integrin signalingrepresent a major mode of action responsible for lunasin’s anticancer activity.
Keywords: Lunasin, Bioactive peptide, Cancer Therapeutic, Integrins, Cell cycle,Non-small cell lung cancer

1. IntroductionNumerous studies over the years have found a stronglinkage of high soy consumption with a number of healthbenefits, including lower rates of cancer. In recent years,it has become clear that at least part of the anticanceractivity of soy is due the presence of the peptide-lunasin. Lunasin is a 43-44-amino acid peptide that is acomponent of the soybean 2S albumin protein that wasinitially shown to cause mitotic arrest and cell death inmammalian cancer cells.1 Recent studies have nowshown that lunasin has the capacity to inhibit the growthof many cancer cell types including breast cancer, coloncancer and lung cancer.2-4 Thus, it is clear that lunasinmay have potential as a therapeutic agent for thetreatment of several deadly cancers.Lunasin has three motifs that may be responsible for itsbiological activity against cancer; 1) a predicted helixdomain homologous to a conserved region ofchromatin-binding proteins, 2) a Arg-Gly-Asp (RGD) celladhesion motif, and 3) a unique polyaspartic-acid tail(Figure 1). It was initially speculated that the RGD celladhesion motif is involved in lunasin internalization intothe cell, and that helix domain and poly-D tail is requiredfor binding with core histone H3 and H4.5 Based onthese hypotheses, the initial proposed mechanism forlunasin action was that lunasin competes with histone

acetyltransferases by binding to deacetylated histones,resulting in an inhibition of histone acetylation and aconcomitant down regulation of cell cycle-relatedprotein expression and the activation of apoptosis.Based on these studies, my laboratory has focused oncharacterizing the effects of lunasin on lung cancer andelucidating its mechanism of action.

Figure 1: Amino-acid sequence and functional motifs oflunasin.
2. Results and DiscussionOur focus on lung cancer is based on the fact that it is theleading cause of cancer-related deaths among both menand women in the United States, and increasingly,around the world. Lung cancer is divided into two types,small cell lung cancer and non-small cell lung cancer(NSCLC); more than 80% of all incidences of lung cancerare NSCLC6. Our initial studies focused on assessing the

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2 Davis et al.: Lunasin inhibits integrin signaling                                  International Journal of Cancer Therapy and Oncology
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© Davis et al. ISSN 2330-4049

effects of Lunasin on established NSCLC cell lines in
vitro. These studies revealed that all the NSCLC linestested were sensitive to lunasin; however, the surprisingfinding was that only one cell line, H661, was sensitivewhen assayed in standard adherent culture conditions.The other cell lines were only sensitive when assayed innon-adherent, anchorage independent conditions usinga colony-formation assay.4 Our subsequent studiesdemonstrated that lunasin was also able to significantlyinhibit tumor growth in a mouse xenograft model ofNSCLC. Lunasin treatment (30 mg/kg body weight)reduced tumor size of subcutaneous tumors initiated byimplanting NSCLC H1299 cells in nude mice by 63%(Figure 2).4

Figure 2: Reduction of NSCLC H1299 tumor grown in vivo.Adapted from McConnell et al.4These findings are very encouraging and showed for thefirst time that lunasin was active against NSCLC. Furtherstudies on the molecular mechanism oflunasin-mediated inhibition of cell proliferation weredone by comparing the responses of NSCLC cells underadherent conditions where line H611 islunasin-sensitive and H1299 cells are resistant. Thesestudies revealed that lunasin’s ability to inhibit H611proliferation was due to the suppression ofphoslphorylation of the retinoblastoma protein and theconcomitant inhibition of cell cycle progression at theG1/S phase transition.4 A summary of these results isdepicted in Figure 3 which identifies key regulatorypoints where lunasin appears to have an effect.

Figure 3: Model for lunasin inhibition of cell cycleprogression. Red and green arrows indicate the activationor inhibition, respectively, of key cell cycle regulatoryproteins.As previously discussed, earlier reports showed lunasininhibits histone acetyltransferases activity under in vitro

condition, and it is well documented that epigeneticchanges involving histone modifications are importantin initiating and maintaining a cancer cell phenotype.7-9Several studies have documented a direct interaction oflunasin with the core histones H3 and H4 in vitro, so weinitiated experiments to see if we could detectinteractions of H3 and H4 in cells. For this, proximityligation assays (PLAs) were used to demonstrate lunasininteracts with histone in vivo using cell lines H661 andH1299 grown in adherent culture conditions. Thelunasin-H3 interaction levels were significantly higher inlunasin-sensitive H661 cells compared to thelunasin-insensitive H1299 cells, whereas lunasin-H4interaction levels were similar in both cell lines.10 Thesehistone interactions were associated with inhibition ofhistone acetylation at H4K8 and H4K12 in the both celllines (Figure 4). Interestingly, H661 exhibited increasedhistone acetylation level at H4K16 compared to H1299,suggesting a role for this histone acetylation mark inlunasin sensitivity.10 Further studies are required tofunctionally test whether lunasin-histone interactionsare required for lunasin’s antiproliferative effects andthe specific epigenetic changes associated with lunasinsensitivity.

Figure 4: Lunasin-induced changes in histone acetylation inNSCLC cells. From Inaba et al.10.Besides affecting histone acetylation, it is possible thatlunasin could also affect integrin signaling through itsRGD domain. Integrins are well known regulators of cellgrowth, migration, survival, and differentiation.11-13Integrins are heterodimeric transmembrane receptorcomposed of two distinct α and β subunits. The integrinsignaling cascade starts with activation by binding ofvarious extracellular matrix proteins such as fibronectin,vitronectin and thrombospondin to the integrinextracellular domain.14, 15 When integrins are inactive,the integrin β subunit cytoplasmic tail forms a saltbridge with the integrin α subunit tail.16, 17 The binding



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of extracellular ligands with the extracellular domaindisrupts α and β subunit cytoplasmic tail associations,triggering binding of activation proteins such as kindlinto integrin β subunit tails and the initiation of furtherdownstream signaling.18-23We tested whether lunasin could affect integrin in twoways. First, we used PLAs to assess the ability of lunasinto interact with integrins, followed by investigationsassessing lunasin effects on downstream signalingevents. We found that lunasin interacted with integrinsubunits α5 and αv in lunasin-sensitive H661 andlunasin-insensitive H1299 cells; however, theinteraction level with integrin αv was significantlyhigher in H661. Based on the differential bindingintensities of lunasin to different integrin subunits, wehypothesize that in H661 cells, lunasin suppresses cellproliferation through binding to integrin vβ3. Weconfirmed that lunasin does indeed bind with vβ3using co-immunoprecipitation assays (Figure 5).Furthermore, lunasin treatment in H661 impairedbinding of the direct effectors ILK, FAK and kindlin tointegrin β1 and β3 cytoplasmic tails, which is theimportant initial step for activation of integrinsignaling.24, 25 A similar disruption of direct effectorinteractions with integrins was not detected in thelunasin-insensitive H1299 cells. To functionally confirmthat the effects of lunasin are mediated by an vsubunit-containing integrin, we used siRNA-mediatedgene silencing to knock out expression of v in H661cells. Although H661 cells with silenced v expressionexhibited reduced proliferation in the absence of lunasin(thus verifying that v is indeed a therapeutic target inthis cell line), treatment with lunasin did not induce anyfurther decrease in proliferation. This represents the

first clear demonstration that lunasin’s ability to inhibitproliferation in NSCLC cells requires an v- containingintegrin.To further assess lunasin effects on integrin signalingand extend our functional studies, we examined theactivation of key integrin signaling components that areknown to ultimately regulate cell proliferation usingWestern blot analyses. These studies revealed thatlunasin treatment reduces integrin signal-regulatedphosphorylation on FAK, Akt and ERK1/2 in H661 butnot in H1299. Taken together, all of these resultsstrongly suggest that in NSCLC cells, lunasin functions asan integrin-signaling antagonist to inhibit cellproliferation. Our current working model describinglunasin’s mechanism of action is shown in Figure 6.

Figure 5: Co-immunoprecipitation (IP) and western blotanalyses of lunasin-vβ3 interactions. NSCLC H661 cellswere treated with 100 µM lunasin for 24 h. Cell lysateswere prepared and immune-precipitates isolated usinganti-lunasin or anti-αvβ3 antibodies. IPs were subjected toimmunoblot analyses using the indicated probe antibodies.

Figure 6: Model for lunasin’s inhibition of cell cycle progression through suppression of integrin signalin g. Red and greenarrows indicate the activation or inhibition, respectively, of key regulatory proteins. Red X’s indicate disruption of keyprotein-protein interactions.



4 Davis et al.: Lunasin inhibits integrin signaling                                  International Journal of Cancer Therapy and Oncology
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© Davis et al. ISSN 2330-4049

3. ConclusionLunasin is an intriguing multifunctional bioactivepeptide that has significant potential to be developedinto an anticancer therapeutic and/or chemopreventionagent. Our studies showed that lunasin has substantialanticancer activity against NSCLC cells both in vitro andin an in vivo mouse xenograft model. Extensivefunctional studies demonstrated that lunasin interactswith αv-containing integrins and likely functions as anintegrin signaling antagonist. We have recently extendedour studies into malignant melanoma and shown similaranticancer effects both in vitro and in vivo for this deadlycancer. Current studies are focused on the furtherdevelopment of lunasin as a therapeutic.
Conflict of interestJI declares that he has no competing interests. KRD islisted as an inventor on two issued patents relating tothe expression and purification of lunasin peptides andmay benefit financially if the technologies described inthese patents are licensed or sold.
AcknowledgementWe thank Owensboro Grain Company (Owensboro, KY)and the Kentucky Soybean Board for their continuedsupport of our research. JI was supported by a JSPSPostdoctoral Fellowship for Research Abroad. Thesefunders had no role in study design, data collection andanalysis, decision to publish, or preparation of themanuscript.
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