The Role of a Natural Amphibian Skin-Based Peptide, Ranatensin, in Pancreatic Cancers Expressing Dopamine D2 Receptors

Abstract

Despite the progress in early diagnostic and available treatments, pancreatic cancer remains one of the deadliest cancers. Therefore, there is an urgent need for novel anticancer agents with a good safety profile, particularly in terms of possible side-effects. Recently dopaminergic receptors have been widely studied as they were proven to play an important role in cancer progression. Although various synthetic compounds are known for their interactions with the dopaminergic system, peptides have recently made a great comeback. This is because peptides are relatively safe, easy to correct in terms of the improvement of their physicochemical and biological properties, and easy to predict. This paper aims to evaluate the anticancer activity of a naturally existing peptide-ranatensin, toward three different pancreatic cancer cell lines. Additionally, since there is no sufficient information confirming the exact character of the interaction between ranatensin and dopaminergic receptors, we provide, for the first time, binding properties of the compound to such receptors.

Keywords: anticancer agents; dopamine receptors; pancreatic cancer; ranatensin.

Figure 1

Binding affinities and G-protein activity in [³⁵S]GTPγS binding assays of RAN. The top panel demonstrates DRD1 (A,B) and DRD2 (C,D) binding affinity of RAN compared to SCH−23390 and spiperone, respectively in [³H]SCH−23390 and [³H]spiperone competition binding assays in rat brain (A,C) and spinal cord (B,D) membrane homogenates. Membranes were incubated with 2 nM [³H]SCH−23390 and 0.25 nM [³H]spiperone. Values are presented as the means ± SEM for at least three experiments performed in duplicate. The bottom panel represents the intrinsic activity E_max of RAN in the absence or presence of the selective DRD1 antagonist SCH-39166 and the selective DRD2 antagonist risperidone in rat brain (left side) and spinal cord (right side) membrane homogenates. The level of basal activity was defined as 100%, and it is demonstrated with a dotted line. Points represent the means ± SEM for at least three experiments that were performed in triplicate. Statistical significances (*** p < 0.001) based on unpaired t-tests were noted for RAN vs. RAN + DRD2 antagonist.

Figure 2

RAN-induced hemolysis. The graph shows the level of hemolysis of RBC treated with increasing concentrations of RAN after 1 and 4 h of treatment. Statistical analysis using two way-ANOVA followed by the Bonferroni correction revealed no significant differences (p > 0.05) between the results obtained.

Figure 3

Representative Western blot images of DRD2 protein expression in cancer and normal cells (A). The quantitative analysis of the expression of DRD2 (mean ± SEM; expressed as a ratio to β-actin) shows different DRD2 expression levels in the tested cells (B). Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparison test. Results were considered statistically significant at * p < 0.05.

Figure 4

The viability of pancreatic cancer and normal cells treated with RAN. The graphs show the differential effect of RAN on tested cells at different timepoints. A–D represents various pancreatic cancer cell lines tested, i.e. (A) PANC-1; (B) BxPC-3; (C) Capan-1; and (D) NHF for normal human fibroblasts. For each line, differences in the viability of RAN-treated cells between three timepoints were compared. Statistical analysis performed using two-way ANOVA followed by Bonferroni post hoc test. Results were considered statistically significant as follows: ** p < 0.01, *** p < 0.005 for 24 h vs. 48 h; ^### p < 0.005 for 24 h vs. 72 h; ^x p < 0.05, ^xx p < 0.01, ^xxx p < 0.005 for 48 h vs. 72 h.

Figure 5

The influence of RAN on cell migration. Wound-healing assays were performed at 0, 24, and 48 h on RAN-treated and untreated PANC-1, BxPC-3, and NHF cells. For each cell line, differences in the migration of RAN-treated cells between three timepoints were compared. Wound area (%) was evaluated by the rate of cells migrating toward the center of the scratched area upon a time using ImageJ™ software. Images from a phase-contrast microscope showing changes in the area covered by the cells at 0, 24, and 48 h after wounding are presented in the Supplementary Materials in Figures S5–S7. Statistical analysis performed using two-way ANOVA followed by Bonferroni post hoc test. The obtained results were statistically significant as follows: *** p < 0.005 for 0 h vs. 24 h; ^### p < 0.005 for 0 h vs. 48 h; ^x p < 0.05, ^xx p < 0.01, ^xxx p < 0.005 for 24 h vs. 48 h, respectively.

Figure 6

The effect of RAN on the viability of cells with and without risperidone-based inhibition of DRD2. A–D represents various pancreatic cancer cell lines, i.e. (A) PANC-1; (B) BxPC-3; (C) Capan-1; and (D) NHF for normal human fibroblasts. Cells with or without DRD2 blockade were treated with RAN for 24 h, and the differences in cell viability (%) were compared. Two-way ANOVA followed by Bonferroni correction was used to determine any statistical differences. Results were considered statistically significant as follows: ** p < 0.01, *** p < 0.005.

Figure 7

The effect of RAN on the migration of risperidone-pretreated cells and cells treated with RAN alone. Graphs present the comparison of migration rate between cells pretreated with risperidone and treated with RAN only for 24 h. Wound area (%) was evaluated as a function of the rate of cells migrating toward the center of the scratched area at a timepoint using ImageJ™ software. Statistical analysis was performed using two-way ANOVA with Bonferroni correction. For * p < 0.05, ** p < 0.01, and *** p < 0.005, results were considered statistically significant. Representative phase-contrast microscope images are presented in the Supplementary Materials in Figures S5–S7 and S14.