The Antiproliferative Effect of Cyclodipeptides from Pseudomonas aeruginosa PAO1 on HeLa Cells Involves Inhibition of Phosphorylation of Akt and S6k Kinases.

Laura Hernández-Padilla,Mario A Moreno-Eutimio,Alma L Díaz-Pérez,Jesús Campos-García,José Moreno-Rodríguez,Luis A Sánchez-Briones,Homero Reyes-De La Cruz,Dolores Vázquez-Rivera,Victor Meza-Carmen

doi:10.3390/molecules22061024

Abstract

Pseudomonas aeruginosa PAO1, a potential pathogen of plants and animals, produces the cyclodipeptides cyclo(l-Pro-l-Tyr), cyclo(l-Pro-l-Phe), and cyclo(l-Pro-l-Val) (PAO1-CDPs), whose effects have been implicated in inhibition of human tumor cell line proliferation. Our purpose was to investigate in depth in the mechanisms of HeLa cell proliferation inhibition by the PAO1-CDPs. The results indicate that PAO1-CDPs, both purified individually and in mixtures, inhibited HeLa cell proliferation by arresting the cell cycle at the G0–G1 transition. The crude PAO1-CDPs mixture promoted cell death in HeLa cells in a dose-dependent manner, showing efficacy similar to that of isolated PAO1-CDPs (LD50 of 60–250 µM) and inducing apoptosis with EC50 between 0.6 and 3.0 µM. Moreover, PAO1-CDPs showed a higher proapoptotic activity (~103–105 fold) than their synthetic analogs did. Subsequently, the PAO1-CDPs affected mitochondrial membrane potential and induced apoptosis by caspase-9-dependent pathway. The mechanism of inhibition of cells proliferation in HeLa cells involves inhibition of phosphorylation of both Akt-S473 and S6k-T389 protein kinases, showing a cyclic behavior of their expression and phosphorylation in a time and concentration-dependent fashion. Taken together our findings indicate that PI3K–Akt–mTOR–S6k signaling pathway blockage is involved in the antiproliferative effect of the PAO1-CDPs.

Highlights

Pseudomonas aeruginosa colonizes several biological environments, such as soil, plants, and animal tissues, being an important opportunistic pathogen in humans, e.g., causing nosocomial infections [1,2].Several mechanisms driving infection in the host have been attributed to the production of toxins, adhesins, siderophores, and a great number of virulence factors
The CDP mixture from the PAO1 strain (PAO1-CDPs) was composed of following proportions: cyclo(L-Pro-L-Tyr) ~25%, cyclo(L-Pro-L-Val) ~25%, cyclo(L-Pro-L-Phe) ~30%, cyclo(L-Pro-L-Leu) ~10%, and other compounds ~10% (Figure S1, Supplementary Material). We used this crude mixture of CDPs isolated from P. aeruginosa PAO1 cultures to evaluate the antiproliferative effect on HeLa and Caco-2 cells [15]
In the context of antiproliferative properties attributed to CDPs, we recently reported that a mixture of CDPs composed of cyclo(L-Pro-L-Tyr), cyclo(L-Pro-L-Val), and cyclo(L-Pro-L-Phe) isolated from the P. aeruginosa PAO1 strain can inhibit the proliferation of human tumor cell lines: HeLa and CaCo-2 [15]

Summary

Introduction

Pseudomonas aeruginosa colonizes several biological environments, such as soil, plants, and animal tissues, being an important opportunistic pathogen in humans, e.g., causing nosocomial infections [1,2].Several mechanisms driving infection in the host have been attributed to the production of toxins, adhesins, siderophores, and a great number of virulence factors. In Staphylococcus aureus, aureusimines A/B, namely, CDPs cyclo(L-Val-L-Tyr) and cyclo(L-Val-L-Phe), respectively, are involved in the regulation of bacterial virulence factors in a murine host [7]. It was reported that CDPs cyclo(L-Leu-L-Pro) and cis-cyclo(L-Phe-L-Pro) isolated from Lactobacillus show antiviral activity against the influenza A (H3N2) virus [8]. Cyclo(L-Phe-L-His) of Aspergillus ustus inhibits the cell cycle in various cancer cell lines [10], whereas cyclo(L-Phe-L-Pro) from L. plantarum induces apoptosis in colon cancer HT-29 cells [11]. Synthetic CDPs such as cyclo(Phe-Pro) induce apoptosis in the HT-29 colon cancer cell line, and cyclo(L-Cys-L-Leu) has a potential for scavenging of free radicals [12]. The molecular mechanisms behind the induction of cell death in cancer cell lines by CDPs involve biological processes such as microtubule polymerization [13]

Methods

Results

Conclusion