Keith R. Davis

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.

Ascorbic Acid and a Cytostatic Inhibitor of Glycolysis Synergistically Induce Apoptosis in Non-Small Cell Lung Cancer Cells

Ascorbic acid (AA) exhibits significant anticancer activity at pharmacologic doses achievable by parenteral administration that have minimal effects on normal cells. Thus, AA has potential uses as a chemotherapeutic agent alone or in combination with other therapeutics that specifically target cancer-cell metabolism. We compared the effects of AA and combinations of AA with the glycolysis inhibitor 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3-PO) on the viability of three non-small cell lung cancer (NSCLC) cell lines to the effects on an immortalized lung epithelial cell line. AA concentrations of 0.5 to 5 mM caused a complete loss of viability in all NSCLC lines compared to a <10% loss of viability in the lung epithelial cell line. Combinations of AA and 3-PO synergistically enhanced cell death in all NSCLC cell lines at concentrations well below the IC50 concentrations for each compound alone. A synergistic interaction was not observed in combination treatments of lung epithelial cells and combination treatments that caused a complete loss of viability in NSCLC cells had modest effects on normal lung cell viability and reactive oxygen species (ROS) levels. Combination treatments induced dramatically higher ROS levels compared to treatment with AA and 3-PO alone in NSCLC cells and combination-induced cell death was inhibited by addition of catalase to the medium. Analyses of DNA fragmentation, poly (ADP-ribose) polymerase cleavage, annexin V-binding, and caspase activity demonstrated that AA-induced cell death is caused via the activation of apoptosis and that the combination treatments caused a synergistic induction of apoptosis. These results demonstrate the effectiveness of AA against NSCLC cells and that combinations of AA with 3-PO synergistically induce apoptosis via a ROS-dependent mechanism. These results support further evaluation of pharmacologic concentrations of AA as an adjuvant treatment for NSCLC and that combination of AA with glycolysis inhibitors may be a promising therapy for the treatment of NSCLC.

Recombinant Protein Expression in Nicotiana

Recombinant protein pharmaceuticals are now widely used in treatment of chronic diseases, and several recombinant protein subunit vaccines are approved for human and veterinary use. With growing demand for complex protein pharmaceuticals, such as monoclonal antibodies, manufacturing capacity is becoming limited. There is increasing need for safe, scalable, and economical alternatives to mammalian cell culture-based manufacturing systems, which require substantial capital investment for new manufacturing facilities. Since a seminal paper reporting immunoglobulin expression in transgenic plants was published in 1989, there have been many technological advances in plant expression systems to the present time where production of proteins in leaf tissues of nonfood crops such as Nicotiana species is considered a viable alternative. In particular, transient expression systems derived from recombinant plant viral vectors offer opportunities for rapid expression screening, construct optimization, and expression scale-up. Extraction of recombinant proteins from Nicotiana leaf tissues can be achieved by collection of secreted protein fractions, or from a total protein extract after grinding the leaves with buffer. After separation from solids, the major purification challenge is contamination with elements of the photosynthetic complex, which can be solved by application of a variety of facile and proven strategies. In conclusion, the technologies required for safe, efficient, scalable manufacture of recombinant proteins in Nicotiana leaf tissues have matured to the point where several products have already been tested in phase I clinical trials and will soon be followed by a rich pipeline of recombinant vaccines, microbicides, and therapeutic proteins.

C4 protein of Beet severe curly top virus is a pathomorphogenetic factor in Arabidopsis.

The Curtovirus C4 protein is required for symptom development during infection of Arabidopsis. Transgenic Arabidopsis plants expressing C4 from either Beet curly top virus or Beet severe curly top virus produced phenotypes that were similar to symptoms seen during infection with wild-type viruses. The pseudosymptoms caused by C4 protein alone were novel to transgenic Arabidopsis and included bumpy trichomes, severe enations, disorientation of vascular bundles and stomata, swelling, callus-like structure formation, and twisted siliques. C4 induced abnormal cell division and altered cell fate in a variety of tissues depending on the C4 expression level. C4 protein expression increased the expression levels of cell-cycle-related genes CYCs, CDKs and PCNA, and suppressed ICK1 and the retinoblastoma-related gene RBR1, resulting in activation of host cell division. These results suggest that the Curtovirus C4 proteins are involved actively in host cell-cycle regulation to recruit host factors for virus replication and symptom development.

Ascorbic acid alleviates toxicity of paclitaxel without interfering with the anticancer efficacy in mice

Paclitaxel is used extensively as a chemotherapeutic agent against a broad range of tumors but often leads to the early termination of treatment due to severe toxic side effects. In this study, we hypothesized that ascorbic acid could reduce the toxic side effects without interfering with the anticancer effect of paclitaxel. To demonstrate this, we examined the effect of the combinational treatment of ascorbic acid and paclitaxel using H1299 (a non-small cell lung cancer cell line) and BALB/c mice implanted with or without sarcoma 180 cancer cells. In H1299 cells, the anticancer effects of the combinational treatment with paclitaxel and ascorbic acid were up to 1.7-foldhigher than those of single-agent paclitaxel treatment. In addition, it was shown that the viability of the HEL299 normal cells was up to 1.6-fold higher with the combinational treatment than with paclitaxel treatment alone. In vivo mouse experiments also showed that mice co-treated with paclitaxel and ascorbic acid did not exhibit the typical side effects induced by paclitaxel, such as a reduction in the numbers of white blood cells and red blood cells and the level of hemoglobin (P < .05). The analysis of cancer-related gene expression by quantitative real-time polymerase chain reaction and immunohistochemistry revealed that the combinational treatment suppressed cancer cell multiplication. Taken together, these results suggest that combinational chemotherapy with ascorbic acid and paclitaxel not only does not block the anticancer effects of paclitaxel but also alleviates the cytotoxicity of paclitaxel in vivo and in vitro.

Lunasin is a novel therapeutic agent for targeting melanoma cancer stem cells

Recent studies provide compelling evidence that melanoma is initiated and maintained by a small population of malignant cells called cancer-initiating cells (CICs) that exhibit stem-cell-like properties. Observations that CICs have a distinct biology when compared to that of the bulk tumor cells and, importantly, are resistant to chemotherapies and radiation, suggest that CICs are involved in invasion, metastasis, and ultimately relapse. Lunasin, a bioactive peptide present in soybean, has both chemopreventive activity and chemotherapeutic activity against multiple cancer types. In this study, we tested the potential of Lunasin to specifically target CICs in melanoma tumor cell populations. In vitro studies using human melanoma cell lines showed that Lunasin treatment decreased the size of a subpopulation of melanoma cells expressing the surrogate CIC marker, Aldehyde Dehydrogenase, concomitant with a reduction in the ability to form colonies in soft agar assays, and reduced tumor growth in mouse xenografts. Similarly, Lunasin inhibited colony formation by isolated melanoma CICs in soft agar and reduced oncosphere formation in vitro and substantially inhibited tumor growth in mouse xenografts. Mechanistic studies revealed that Lunasin treatment of isolated melanoma CICs induced expression of the melanocyte-associated differentiation markers Tyrosinase and Microphthalmia-associated Transcription Factor concomitant with reduced expression of the stemness factor NANOG. These findings document for the first time that Lunasin has significant therapeutic activity against melanoma by specifically targeting melanoma CICs, and inducing a more differentiated, non-CIC phenotype. Thus, Lunasin may represent a novel therapeutic option for both chemoresistant and advanced metastatic melanoma management.

The Arabidopsis thaliana homeobox gene ATHB12 is involved in symptom development caused by geminivirus infection.

Background Geminiviruses are single-stranded DNA viruses that infect a number of monocotyledonous and dicotyledonous plants. Arabidopsis is susceptible to infection with the Curtovirus, Beet severe curly top virus (BSCTV). Infection of Arabidopsis with BSCTV causes severe symptoms characterized by stunting, leaf curling, and the development of abnormal inflorescence and root structures. BSCTV-induced symptom development requires the virus-encoded C4 protein which is thought to interact with specific plant-host proteins and disrupt signaling pathways important for controlling cell division and development. Very little is known about the specific plant regulatory factors that participate in BSCTV-induced symptom development. This study was conducted to identify specific transcription factors that are induced by BSCTV infection. Methodology/Principal Findings Arabidopsis plants were inoculated with BSCTV and the induction of specific transcription factors was monitored using quantitative real-time polymerase chain reaction assays. We found that the ATHB12 and ATHB7 genes, members of the homeodomain-leucine zipper family of transcription factors previously shown to be induced by abscisic acid and water stress, are induced in symptomatic tissues of Arabidopsis inoculated with BSCTV. ATHB12 expression is correlated with an array of morphological abnormalities including leaf curling, stunting, and callus-like structures in infected Arabidopsis. Inoculation of plants with a BSCTV mutant with a defective c4 gene failed to induce ATHB12. Transgenic plants expressing the BSCTV C4 gene exhibited increased ATHB12 expression whereas BSCTV-infected ATHB12 knock-down plants developed milder symptoms and had lower ATHB12 expression compared to the wild-type plants. Reporter gene studies demonstrated that the ATHB12 promoter was responsive to BSCTV infection and the highest expression levels were observed in symptomatic tissues where cell cycle genes also were induced. Conclusions/Significance These results suggest that ATHB7 and ATHB12 may play an important role in the activation of the abnormal cell division associated with symptom development during geminivirus infection.

The soy-derived peptide Lunasin inhibits invasive potential of melanoma initiating cells

Lunasin is a 44 amino acid peptide with multiple functional domains including an aspartic acid tail, an RGD domain, and a chromatin-binding helical domain. We recently showed that Lunasin induced a phenotype switch of cancer initiating cells (CIC) out of the stem compartment by inducing melanocyte-associated differentiation markers while simultaneously reducing stem-cell-associated transcription factors. In the present study, we advance the hypothesis that Lunasin can reduce pools of melanoma cells with stem cell-like properties, and demonstrate that Lunasin treatment effectively inhibits the invasive potential of CICs in vitro as well as in vivo in a mouse experimental metastasis model. Mice receiving Lunasin treatment had significantly reduced pulmonary colonization after injection of highly metastatic B16-F10 melanoma cells compared to mice in the control group. Mechanistic studies demonstrate that Lunasin reduced activating phosphorylations of the intracellular kinases FAK and AKT as well as reduced histone acetylation of lysine residues in H3 and H4 histones. Using peptides with mutated activity domains, we functionally demonstrated that the RGD domain is necessary for Lunasin uptake and its ability to inhibit oncosphere formation by CICs, thus confirming that Lunasins ability to affect CICs is at least in part due to the suppression of integrin signaling. Our studies suggest that Lunasin represents a unique anticancer agent that could be developed to help prevent metastasis and patient relapse by reducing the activity of CICs which are known to be resistant to current chemotherapies.

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

Abstract 602: Ascorbic acid combined with a cytostatic inhibitor of glycolysis synergistically induces apoptosis in non-small lung cancer cells.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Purpose An exceptional characteristic of many cancer cells is increased glucose uptake and elevated glycolysis with a concomitant reduction in oxidative respiration, also known as the Warburg effect. The increase in basal levels of reactive oxygen species (primarily, H2O2) and their increased dependence on glycolysis for their survival make tumor cells more susceptible than normal cells to treatment with a combination of pro-oxidant agents and chemotherapy. This common feature of tumor cells has been targeted for the development of new cancer therapeutics. Ascorbic acid (AA) has been shown to have cancer curative potential, at clinically achievable doses that have minimal or no toxic effects on normal cells. We have previously shown that some cancer cell lines are significantly more sensitive to treatment with ascorbic acid at concentrations where it may function as a pro-oxidant. In the present case, we investigated the effects of AA alone or in combinations of AA with the glycolysis inhibitor 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3-PO) on the viability of three non-small lung cancer (NSCLC) cell lines vs immortalized lung epithelial cell line. Results Cell viability assays showed that combined treatment of AA and 3-PO caused a synergistic increase in cell death in all the NSCLC cell lines at concentrations well below the IC50 concentrations for each compound alone, while no synergistic cytotoxic effect was observed in immortalized lung epithelial cells. Key markers such as DNA fragmentation, PARP cleavage, annexin-V binding, and caspase activity indicated that AA-induced cell death is caused via the activation of apoptosis. Metabolomic analysis revealed a decrease in NAD/NADH in the combination treatment that correlated well with the synergistic cell death observed in the viability results. The importance of NAD/NADH depletion in the synergistic response was confirmed by rescue experiments showing addition of NAD to the culture protected cells from the combination treatment. The major significance of these studies is that, we show for the first time that AA selectively synergizes with a glycolytic inhibitor in killing NSCLC cells and that pharmacologic AA treatment (either alone or combined with any other chemotherapeutic agent or glycolytic inhibitors used in the treatment of cancer) may be a promising therapy for non-small lung cancer and other challenging to-treat cancers. Citation Format: Saleha B. Vuyyuri, Jacob Rinkinen, Erin Worden, Keith R. Davis. Ascorbic acid combined with a cytostatic inhibitor of glycolysis synergistically induces apoptosis in non-small lung cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 602. doi:10.1158/1538-7445.AM2013-602