METHODS AND EXPERIMENTAL DESIGN:Peptide Synthesis: All analogs of CDT were synthesized by solid phase peptide synthesis on a PS3 Automated Peptide Synthesizer by Protein Technologies

Figure 2. Schematic Representation of Solid-Phase Peptide Synthesis

A549 Cancer Cell Culture: Cells were removed from storage in liquid nitrogen and diluted to 12,500 cells per mL using culture media. These cells were incubated for 1-2 days in a 96-well plate.

ASSAYS to show Anticancer Properties of CDT Analogs:Cell Viability Assay (MTT): The tetrazolium dye MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) can be reduced to its purple formazan by cellular oxidoreductase enzymes which are only present in viable cells. Where there is more purple color present, there are more viable cells. The absorbance at 550 nm was obtained using a spectrophotometer. This absorbance was converted to percent cell viability relative to the untreated control.

Peptide Dosing: CDT and its analogs were dissolved in 5% DMSO. Cells were dosed with peptide concentrations ranging rom 4.2 – 540 mM. The negative control only contained culture media, and the positive control contained butyric acid. Another control contained only 5% DMSO, to ensure that DMSO did not kill cells.

Western Blotting: A549 cells treated with 656 μM CDT (approximate IC50 of CDT), purified Humanin and control were grown in culture media and harvested after 48h. The cells were resuspended in 25 μL SSB (staining solution) and 20 μL were loaded on the gel. ERK antibodies, catalog number sc-292838, and Phospho-ERK antibodies ,catalog number sc-2020 were used

The Potential Anti-Cancer Effects of the Antimicrobial Peptide CDT and its Analogs on A549 Lung Cancer CellsMurali Jujjavarapu, Anna Eitel, Deborah Heyl-Clegg, Hedeel Evans, Jeffrey W.Guthrie

Department of Chemistry, Eastern Michigan UniversityYpsilanti, MI 48197 U.S.A

ABSTRACTIn spite of incredible advances in cancer treatment, new anticancer agents which have different modes of action are continually sought. Recently, it was noted that some antimicrobial peptides also show anti-cancer effects. Tachyplesin is an antimicrobial peptide from horseshoe crab that exhibits anti-bacterial activity by destroying cell membranes; it has also been shown to kill cancer cells.. Our lab has studied a linear version of this peptide, Cysteine deleted Tachyphlesin (CDT), as an antibiotic, but to date it has not been tested for effects against cancer cells. In order to study the potential anti-cancer effects of this peptide, synthesized through the solid-phase technique, we are testing CDT along with its analogs against cultured A549 lung cancer cells. In addition, we perform MTT assay to understand their cytotoxicity on cancer cells. Results indicate that the CDT and its analogs have indeed kill cancer cells. Based on the data available we construct graph showing IC50 of each analog. We then compare these results with western blotting technique.

INTRODUCTIONCancer cells are very harmful to the body because they divide uncontrollably and are able to attack the rest of the body through the bloodstream. Many key biological pathways are affected by it. ERK pathway (extracellular signal-regulated kinases)  is a chain of proteins in the cell that communicates a signal from a receptor on the surface of the cell to the DNA in the nucleus of the cell. This pathway is affected when one of the proteins is mutated and results in cancer development. Peptide drugs are an innovative way of treating cancer. Antimicrobial peptides are potent, broad spectrum antibiotics which represent potential novel therapeutic agents. Tachyplesin, a cyclic peptide, is one of the antimicrobial peptides that was isolated from the horseshoe crab, which has shown anti-cancer effects. In our laboratory we study Cysteine-Deleted- Tachyplesin (CDT), which is a linear analog of Tachyplesin. Cancer cell membranes contain negatively charged molecules such as phosphatydyl-serine, sialic acid, and heparin sulfate. The positively charged arginine and lysine in CDT are attracted to the negatively charged cancer cells, disrupting the cancer cell membrane and causing apoptosis. Normal mammalian cell membranes, however, consist largely of zwitterionic phospholipids, giving them an overall neutral charge. This allows CDT to be selective against only cancer cells, and not healthy cells.

GOALS:To synthesize and purify CDT analogs and test them for anti-cancer effects on A-549 lung cancer cells. • To determine the percent of viable cancer cells through performing MTT assay• To compare The ERK and Phospho ERK of CDT treated A549 cancer cells with

that of control sample and humanin peptide.

LITERATURE CITED:1. Ramamoorthy, A., Thennarasu, S., Tan, A., Gottipati, K., Sreekumar, S., Heyl, D.L., An, F., Shelburne, C.E. Biochemistry (2006) 45:6529-6540.2. Deborah Heyl, Yeji Park, Jennifer Garvey, Rebecca Newman, and Yllka Vladaj (2015) In Proceedings of the 24th American Peptide Symposium), Michal Lebl, Ved Srivastava, and Andrei Yudin, eds., 66-68.3. Saravanan, R., Mohonram, H., Joshi, M., Domadia, P.N., Torres, J., Ruedl, C., Bhattacharjya, S. Biochimica et Biophysica Acta (BBA) – Biomembranes (2012) 1818 (7): 1613-1624.4. Schreier, S., Malheiros, S., De Paula, E., Biochim Biophys Acta (2000) 1508: 210-234. 5. Wood S.J., Park Y.A., Kanneganti N.P., Mukkisa H.R., Crisman L.L., Davis S.E., Vandenbosch J.L., Scaglione, J., Heyl, D.L. Int. J. Peptide Res. Ther. (2014) 20 (4): 519-530.

Figure 1. Two different models of peptide permeation of a membrane: 1) The “carpet” method. 2) The “Barrel-Stave method

DISCUSSION All-D CDT was the most potent peptide, with an IC50 of 24.37 µM of peptide.

oThis was considerably more potent than the other analogs of CDT as the second lowest IC50 was about 100 µM. CDT itself and its reverse sequence had lower activity (higher IC50 values).

A previous study examining the minimum inhibitory concentrations (MIC) of CDT analogs on E. coli and S. aureus bacteria found that All-D CDT had a lower MIC in both bacteria and a higher selectivity for bacterial over mammalian cells (RBC) than other analogs. oThe all-D CDT analog has shown increased potency against bacteria as well as lung cancer

cells, and is not detrimental to normal mammalian cells based on hemolytic assays (data not shown).

The all-D analog had the same sequence as CDT, but had a different stereoisomeric configuration. oThe D-amino acids could have caused the peptide to have longer action in the live cancer cells

because cellular enzymes are selective based on chirality.• Western blotting: CDT treated A549 cancer cells shows less phospho-ERK when compared to

that of humanin and control which indicates that the ERK pathway may be affected by CDT, influencing cell death.

ACKNOWLEDGMENTSGraduate research student Daniel Esckilsen, Dr. R.A. Armitage for confirming peptides via mass spectroscopy, and the Seller's Fund/Provost's Research Support Award for funding.

Total ERK

P-ERK

1 2 3

Amino acids: N-α Fluorenylmethyloxycarbonyl (Fmoc) and side chain protected (Anaspec), activated as carboxylic esters by HBTU (Anaspec)

Activator: 0.4M N,N-Diisopropylethylamine in DMF (VWR) Deprotectant: 20% piperidine in DMF (VWR) Resin: p-methylbenzhydrylamine (MBHA) (0.7mmol/g) (Bachem)Cleavage and Purification: (Solvents and reagents from VWR, Fisher, and Sigma)Peptides were cleaved using 9 mL trifluoroacetic acid, 0.5 mL distilled water, 0.5 mL phenol and 200 microliters triisopropylsilane

Purification and Peptide Analysis:Purification was accomplished through reversed-phase high-performance liquid chromatography (RP-HPLC)Identification was confirmed by electrospray mass spectrometry.

Figure 7 .The ERK and Phospho ERK results ofA549 cells treated with 1) 656 μM CDT (approximate IC50 of CDT), 2) purified Humanin and 3) Control

0 100 200 300 400 500 600020406080

100120

Figure 3. The percent viability of A549 lung cancer cells when dosed with varying concentrations of CDT.

Concentration of Peptide (µM)

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Figure 4. The percent viability of A549 lung cancer cells when dosed with varying concentrations of

Reverse CDT.

Concentration of Peptide (µM)

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Figure 5. The percent viability of A-549 lung cancer cells when dosed with varying concentrations of All-

D CDT.

Concentration of Pepide (µM)

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Figure 6. The percent viability of A549 lung cancer cells when dosed with varying concentrations of All-D Re-

verse CDT.

Concentration of Peptide (µM)

Perc

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Sample Name Sequence IC50 (µM)

CDT KWFRVYRGIYRRR 656.1

Reverse CDT RRRYIGRYVRFWK 314.5

All-D CDT kwfrvyrgiyrrr 24.37

All-D Reverse CDT rrryigryvrfwk 96.99

Table 1. The name, sequence, and half maximal inhibitory concentration (IC50) of CDT and three of its analogs found by dosing A549 cells with varying concentrations of each peptide.

RESULTS: