Elizabeth J. McConnell

Scalable Purification and Characterization of the Anticancer Lunasin Peptide from Soybean

Lunasin is a peptide derived from the soybean 2S albumin seed protein that has both anticancer and anti-inflammatory activities. Large-scale animal studies and human clinical trials to determine the efficacy of lunasin in vivo have been hampered by the cost of synthetic lunasin and the lack of a method for obtaining gram quantities of highly purified lunasin from plant sources. The goal of this study was to develop a large-scale method to generate highly purified lunasin from defatted soy flour. A scalable method was developed that utilizes the sequential application of anion-exchange chromatography, ultrafiltration, and reversed-phase chromatography. This method generates lunasin preparations of >99% purity with a yield of 442 mg/kg defatted soy flour. Mass spectrometry of the purified lunasin revealed that the peptide is 44 amino acids in length and represents the original published sequence of lunasin with an additional C-terminal asparagine residue. Histone-binding assays demonstrated that the biological activity of the purified lunasin was similar to that of synthetic lunasin. This study provides a robust method for purifying commercial-scale quantities of biologically-active lunasin and clearly identifies the predominant form of lunasin in soy flour. This method will greatly facilitate the development of lunasin as a potential nutraceutical or therapeutic anticancer agent.

TNF potentiates anticancer activity of bortezomib (Velcade®) through reduced expression of proteasome subunits and dysregulation of unfolded protein response

Bortezomib (Velcade®) exploits proteasome inhibition as a unique mechanism of anticancer activity. The effectiveness of bortezomib is, however, limited, therefore, the search for therapeutic regimens combining bortezomib with other agents. In the present work we demonstrate enhanced anticancer activity of bortezomib by its combination with tumor necrosis factor (TNF) in the experimental model of C‐26 colon carcinoma in mice. This interaction likely relies on the induction of a dysregulated response to ER stress, leading to apoptosis of cancer cells, evidenced by caspase‐3 cleavage, p53 accumulation as well as increased SAPK/JNK phosphorylation. ER stress induced by the combination of TNF and bortezomib is corroborated by upregulation of BiP, PDI and calnexin as well as cleavage of caspase‐12; however, in contrast to the classic pathway, it is also associated with decreased phosphorylation of eIF2α and prevention of XBP‐1 splicing. TNF prevented the upregulation of Hsp27 induced by bortezomib, which may contribute to enhanced ER stress. Moreover, TNF interfered with bortezomib‐induced upregulation of distinct subunits of the 26S proteasome. Bortezomib concentration used in this study was not sufficient to prevent TNF from inducing nuclear translocation of p65/RelA; however, the combination of both agents reduced total p65/RelA levels. Combined treatment of tumor‐bearing mice with bortezomib and TNF not only inhibited tumor growth but also significantly prolonged animal survival. Therefore, combination of bortezomib with TNF is an attractive option for further clinical studies.

Decreased ER-associated degradation of α-TCR induced by Grp78 depletion with the SubAB cytotoxin

HeLa cells stably expressing the alpha chain of T-cell receptor (alphaTCR), a model substrate of ER-associated degradation (ERAD), were used to analyze the effects of BiP/Grp78 depletion by the SubAB cytotoxin. SubAB induced XBP1 splicing, followed by JNK phosphorylation, eIF2alpha phosphorylation, upregulation of ATF3/4 and partial ATF6 cleavage. Other markers of ER stress, including elements of ERAD pathway, as well as markers of cytoplasmic stress, were not induced. SubAB treatment decreased absolute levels of alphaTCR, which was caused by inhibition of protein synthesis. At the same time, the half-life of alphaTCR was extended almost fourfold from 70 min to 210 min, suggesting that BiP normally facilitates ERAD. Depletion of p97/VCP partially rescued SubAB-induced depletion of alphaTCR, confirming the role of VCP in ERAD of alphaTCR. It therefore appears that ERAD of alphaTCR is driven by at least two different ATP-ase systems located at two sides of the ER membrane, BiP located on the lumenal side, while p97/VCP on the cytoplasmic side. While SubAB altered cell morphology by inducing cytoplasm vacuolization and accumulation of lipid droplets, caspase activation was partial and subsided after prolonged incubation. Expression of CHOP/GADD153 occurred only after prolonged incubation and was not associated with apoptosis.

Inhibition of calmodulin-stimulated (Ca2+ + Mg2+)-ATPase activity by dimethyl sulfoxide

Membrane-bound (Ca2+ + Mg2+)-ATPase activity from human erythrocyte white ghosts in the calmodulin-activated state was inhibited by DMSO in concentrations of 3% (v/v) and above. At 10%, DMSO inhibited calmodulin activation by 47.7%, while basal, calmodulin-independent (Ca2+ + Mg2+)-ATPase and (Mg2+)-ATPase activity remained unaffected. (Na+ + K+)-ATPase activity was also reduced but exhibited a greater IC50. Concentration-effect analyses showed the inhibition by 10% DMSO to be a reversible, non-competitive effect with regard to calmodulin, Ca2+, and substrate activation. Calmodulin-stimulated processes may be more susceptible to inhibition by DMSO than related enzymatic catalysis, and thus may help explain the multitude of reported cellular events caused by the solvent. Furthermore, DMSO affected membrane-associated enzymatic mechanisms opposite to those reported for purified enzyme outside its native membrane environment.

Lunasin Sensitivity in Non-Small Cell Lung Cancer Cells Is Linked to Suppression of Integrin Signaling and Changes in Histone Acetylation

Lunasin is a plant derived bioactive peptide with both cancer chemopreventive and therapeutic activity. We recently showed lunasin inhibits non-small cell lung cancer (NSCLC) cell proliferation in a cell-line-specific manner. We now compared the effects of lunasin treatment of lunasin-sensitive (H661) and lunasin-insensitive (H1299) NSCLC cells with respect to lunasin uptake, histone acetylation and integrin signaling. Both cell lines exhibited changes in histone acetylation, with H661 cells showing a unique increase in H4K16 acetylation. Proximity ligation assays demonstrated lunasin interacted with integrins containing αv, α5, β1 and β3 subunits to a larger extent in the H661 compared to H1299 cells. Moreover, lunasin specifically disrupted the interaction of β1 and β3 subunits with the downstream signaling components phosphorylated Focal Adhesion Kinase (pFAK), Kindlin and Intergrin Linked Kinase in H661 cells. Immunoblot analyses demonstrated lunasin treatment of H661 resulted in reduced levels of pFAK, phosphorylated Akt and phosphorylated ERK1/2 whereas no changes were observed in H1299 cells. Silencing of αv expression in H661 cells confirmed signaling through integrins containing αv is essential for proliferation. Moreover, lunasin was unable to further inhibit proliferation in αv-silenced H661 cells. This indicates antagonism of integrin signaling via αv-containing integrins is an important component of lunasin’s mechanism of action.

Analysis of Npl4 deletion mutants in mammalian cells unravels new Ufd1-interacting motifs and suggests a regulatory role of Npl4 in ERAD

Npl4 is a 67 kDa protein forming a stable heterodimer with Ufd1, which in turn binds the ubiquitous p97/VCP ATPase. According to a widely accepted model, VCP(Ufd1-Npl4) promotes the retrotranslocation of emerging ER proteins, their ubiquitination by associated ligases, and handling to the 26S proteasome for degradation in a process known as ERAD (ER-associated degradation). Using a series of Npl4 deletion mutants we have revealed that the binding of Ufd1 to Npl4 is mediated by two regions: a conserved stretch of amino acids from 113 to 255 within the zf-Npl4 domain and by the Npl4 homology domain between amino acids 263 and 344. Within the first region, we have identified two discrete subdomains: one involved in Ufd1 binding and one regulating VCP binding. Expression of any one of the mutants failed to induce any changes in the morphology of the ER or Golgi compartments. Moreover, we have observed that overexpression of all the analyzed mutants induced mild ER stress, as evidenced by increased Grp74/BiP expression without associated XBP1 splicing or induction of apoptosis. Surprisingly, we have not observed any accumulation of the typical ERAD substrate alphaTCR. This favors the model where the Ufd1-Npl4 dimer forms a regulatory gate at the exit from the retrotranslocone, rather than actively promoting retrotranslocation like the p97VCP ATPase.

Intracellular Ca 2+ Homeostatic Regulation and 4-Hydroxynonenal-Induced Aortic Endothelial Dysfunction

The aldehydic lipid peroxidation product 4-hydroxynonenal (HNE) is known to compromise erythrocyte passive Ca2+ permeability and to irreversibly inhibit the plasma membrane (Ca2+ + Mg2+)-ATPase and Ca2+-transport. To measure the effects of HNE on passive and active Ca2+ transport in endothelial cells, we first characterized 45Ca2+ uptake and efflux in cultured porcine aortic endothelial cells (PAEC). PAEC exchanged 45Ca2+ to a cumulative near-isotopic equilibrium of about 4.5 pmole 45Ca2+/10(6) cells in 120 min at 37 degrees C. This Ca2+ pool was diminished by thapsigargin, cyclopiazonic acid, oligomycin B, and sodium azide. In contrast, ouabain enhanced Ca2+ uptake capacity from 5.17 to 5.77 pmole/10(6) cells. Accumulated 45Ca2+ was extruded at rate of 8.7 fmole 45Ca2+/10(6) cells/min or shunted rapidly by the ionophore A23187. HNE increased total 45Ca2+ accumulation in a time- and concentration-dependent manner by as much as 562% with an EC50 of 64.0 wM. Concomitant morphological analysis of PAEC revealed vacuolization, nuclear swelling, cell shrinking, and cell detachment. Initial structural changes, such as vacuolization, began well before any changes in Ca2+ accumulation were observed. These functional and morphological changes indicate that HNE significantly increases intracellular Ca2+ accumulation in vascular endothelium, which may explain the cytotoxic effects associated with HNE exposure and provide further evidence that atherogenic effects of HNE may, in part, be caused by disturbances in Ca2+ homeostasis.

Abstract 3850: Production of recombinant lunasin peptides with enhanced anticancer activity using transient expression in tobacco

Epidemiological observations suggest a correlation between high levels of soybean product consumption and lowered incidence and mortality due to prostate, breast and colon cancer. Lunasin, a 43-44 amino acid peptide derived from soybean, has been implicated as a significant source of this anticancer activity. Lunasin contains nine consecutive aspartic acid residues at the C-terminus that bind to hypoacetlyated core histones, a minimal internal RGD cell adhesion motif and a helical region exhibiting structural homology to conserved sequences of chromatin binding proteins. Initial studies demonstrated that lunasin can prevent the transformation of mammalian cells by chemical carcinogens or viral oncogenes, and inhibits chemically-induced tumors in a mouse skin cancer model. These results suggest that lunasin may be useful as a cancer chemoprevention agent. More recent studies have demonstrated that lunasin can inhibit the proliferation of several established human cancer cell lines in vitro and in vivo, suggesting that lunasin may also be useful as a cancer therapeutic. Lunasin9s anticancer effects are currently limited to specific cancer cell types and require 10-100 µM concentrations of peptide. We have now established a transient expression system based on the Tobacco Mosaic Virus vector, Geneware®, for large-scale production of modified forms of recombinant lunasin in tobacco. Our initial studies revealed that we could not detect any significant protein accumulation using constructs expressing lunasin peptides alone. We modified our strategy by expressing lunasin peptides as C-terminal fusions to green fluorescent protein (GFP) that includes a linker sequence containing a thrombin cleavage site. We have demonstrated that this system can produce GFP-lunasin at levels >100 mg/kg fresh weight tissue and demonstrated that the lunasin peptide can be recovered by proteolytic cleavage with thrombin. We have used this system to produce a modified form of lunasin that contains an N-terminal RGD domain sequence that preferentially binds to the α v β 6 integrin that is highly expressed in a number of epithelial-derived carcinomas. In vitro studies using cancer cell lines expressing the α v β 6 integrin demonstrate that the RGD-lunasin peptide is >10-fold more active in inhibiting cancer cell proliferation than the natural form of lunasin. Moreover, RGD-lunasin inhibits the proliferation of some cancer cell lines that are insensitive to 100 µM lunasin. These results demonstrate the feasibility of utilizing plant-based expression to produce more efficacious forms of lunasin that are targeted specifically to cancer cells and indicate that RGD lunasin may be useful as a cancer therapeutic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3850. doi:1538-7445.AM2012-3850