The minimum element of a synthetic peptide required to block prostate tumor cell migration

Abstract

Human prostate tumor cell invasion and metastasis are dependent in part on cell adhesion to extracellular matrix proteins and cell migration. We previously identified a synthetic D-amino acid tumor cell adhesion peptide called HYD1 (kikmviswkg) that supported adhesion of tumor cells derived from breast, prostate, ovary and pancreas tissue. Alanine substitution analysis and a peptide deletion strategy were used to determine the minimal element of HYD1 necessary for bioactivity in a prostate cancer cell line called PC3N. Bioactivity was measured by assays of cell adhesion, migration and ERK signaling. The most potent element of HYD1 necessary to support cell adhesion was kmvixw, the block to migration required xkmviswxx and activation of ERK signaling required ikmviswxx. The shortest sequence active in all three assays was iswkg. The HYD1 peptide contains overlapping elements required for adhesion, blocking migration and the activation of ERK signaling. These linear peptide sequences provide the starting point for development of novel compounds to target cancer cell adhesion and migration.

Figure 1

The minimal element mediating cell adhesion to immobilized HYD1 is kmvixw. The ability of PC3N cells to adhere within 60 minutes to either immobilized variants of HYD1 containing systematic alanine substitution (A) at the indicated residues or (B) deletion variants of the HYD1 peptide. The residue that was substituted by alanine (a) is as indicated. Similarly, the sequence of the variant forms in the deletion analysis is as indicated. The amount of bound cells was estimated by releasing the cell bound crystal violet dye using 0.1 M sodium citrate and reading the absorbance at 562 nm. All determinations were done in triplicate and the error bars represent one standard deviation about the mean. The data obtained from both the alanine substitution and deletion analysis is collated in (C).

Figure 2

The minimal element to block cell migration is xkmviswxx. (A) PC3N cells were grown to a monolayer on laminin 322 and cell migration was induced by scratching the monolayer surface. The cells were incubated with the indicated alanine substitution sequences of HYD1 at a concentration of 50 μM in the presence of 1% FBS for 12 hours following the scratch. (B) PC3N cells were grown to a monolayer on laminin 322 and cell migration was induced by scratching the monolayer surface. The cells were incubated with the indicated deletion forms of the HYD1 sequences at a concentration of 50 μM in the presence of 1% FBS for 12 hours following the scratch. In both (A and B), the data represent the percent of cells in a microscopic field migrating into the scratch. Three fields were analyzed per sample and the error bars represent one standard deviation about the mean. The data obtained from both the alanine substitution and deletion analysis is collated in (C).

Figure 3

The minimal element to activate ERK signaling is ikmviswxx. (A) PC3N cells were placed on laminin-5 coated tissue culture plates in the presence of serum-free media for 1 hour followed by the addition of HYD1 (kikmviswkg), alanine-substitution mutants of HYD1, or a scrambled version of HYD1, HYDS (wiksmkivkg) at a concentration of 50μM in serum-free media for 10 minutes. (B) PC3N cells were placed on laminin 322 coated tissue culture plates in the presence of serum-free media for 1 hour followed by the addition of HYD1 (kikmviswkg), the indicated deletion forms of HYD1, or HYDS (wiksmkivkg) at a concentration of 50μM in serum-free media for 10 minutes. In both (A and B), cell lysates were analyzed for phosphorylation of ERK Thr-202/Tyr-204. Blots were stripped and reprobed for total ERK. The data obtained from both the alanine substitution and deletion analysis is collated in (C).

Figure 4

The optimal sequences for cell adhesion, ERK signaling and blocking cell migration. The results indicating the minimal sequences from Figures 1, 2 and 3 are aligned for comparative purposes.