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. 2007 Oct 3;2(10):e964.
doi: 10.1371/journal.pone.0000964.

Novel decapeptides that bind avidly and deliver radioisotope to colon cancer cells

John M Abraham  1 , Fumiaki Sato, Yulan Cheng, Bogdan Paun, Takatsugu Kan, Alexandru Olaru, Zhe Jin, Jian Yang, Rachana Agarwal, Stefan David, James P Hamilton, Tetsuo Ito, Yuriko Mori, Stephen J Meltzer
Affiliations

Novel decapeptides that bind avidly and deliver radioisotope to colon cancer cells

John M Abraham et al. PLoS One. .

Abstract

Background: The rapidly growing field of targeted tumor therapy often utilizes an antibody, sometimes tagged with a tumor-ablating material such as radioisotope, directed against a specific molecule.

Methodology/principal findings: This report describes the discovery of nine novel decapeptides which can be radioactively labeled, bind to, and deliver (32)P to colon cancer cells. The decapeptides vary from one another by one to three amino acids and demonstrate vastly different binding abilities. The most avidly binding decapeptide can permanently deliver very high levels of radioisotope to the adenocarcinoma cancer cell lines at an efficiency 35 to 150 times greater than to a variety of other cell types, including cell lines derived from other types of cancer or from normal tissue.

Conclusions/significance: This experimental approach represents a new example of a strategy, termed peptide binding therapy, for the potential treatment of colorectal and other adenocarcinomas.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Schematic diagram of experimental approach.
A bacterial recombinant expression system produced various gluthathione-S-transferase decapeptide fusion proteins which were bound to gluthatione and labeled with 32P utilizing protein kinase A. After washing, the labeled decapeptides were recovered after thrombin digestion and incubated at various times with several different cell lines.
Figure 2
Figure 2. Levels of binding of decapeptide MA5 to eighteen different cell lines.
The 32P labeled decapeptide MA5 was incubated for two hours with 10,000 cells, washed three times, and the radioactive counts of the cells determined by scintillation counting. Seven cell lines demonstrated avid binding of MA5 and are shown as bar graphs of the mean and one standard deviation in triplicate wells. The remaining eleven cell lines, along with one blank well averaged only 365 cpm. These values are so small as to not be visible at the scale used in this figure. Further information on the individual cell lines is provided in the Supplemental Information.
Figure 3
Figure 3. Relative levels of binding of nine 32P-labeled decapeptide variants.
(A) Nine 32P-labeled different decapeptides, varying from one another by only one to three amino acids, were incubated with Caco-2 cells for two hours, the cells washed three times, and counts remaining bound to the cells are shown as a percentage of the total amount of counts for each decapeptide used. Amino acid substitutions for each variant (relative to MA1) are underlined and bolded. (B) The variants, MA1, MA4, and MA5 were incubated with Caco-2 cells for intervals varying from five minutes to two hours, washed, the adherent cells dissolved in gel loading buffer and an aliquot run on a 10%–20% gradient polyacrylamide-SDS gel. The three lanes marked “24h” (lanes 5, 10, and 15) were incubated with the respective labeled decapeptides (MA1, MA4, MA5) for two hours, washed, and the cells incubated with complete medium for 24 hours. The cells were treated as described for the other lanes of this figure.
Figure 4
Figure 4. The majority of the 32P-labeled decapeptide MA5-bound molecules are released from Caco-2 cells.
(A) The 32P-labeled decapeptide MA5 was incubated for two hours with seven different cell lines, the cells were washed, and complete medium was added. Following a 24 hour incubation, the number of counts per minute released into the medium (R) as well as the number of counts remaining bound to the cells (B) were determined. Each bar shows the mean and one standard deviation of triplicates wells. (B) Time course for the release and retention of the 32P-labeled decapeptide MA5. MA5 was incubated for two hours with Caco-2 cells, the cells washed, and the cpm released (dashed line) as well as remaining bound (solid line) to the cells determined for time intervals post-washing. Each point shows the mean plus/minus one standard deviation of triplicate determinations. C) Radioactive well contents described as bound (solid line) in Figure 4B were run on a 16.5% polyacrylamide-SDS gel and exposed to film. Immediately after washing (i.e., at 0 hours), the majority of the counts were visualized as 32P-peptide. Over the next 48 hours, the peptide counts greatly diminished, with the majority of bound radioactivity incorporated into cellular proteins. (D) Aliquots of medium containing the released (dotted line) 32P-peptide MA5 were assayed at time intervals after washing, as described in Figure 4B. As time progressed, more of the 32P-peptide was released, reaching a plateau by 24 hours after washing.
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