Antitumor efficacy of Kisspeptin in human malignant mesothelioma cells

Abstract

Purpose: Kisspeptin signaling, via its receptors GPR54, could be an essential players in the inhibition of mesothelioma progression, invasion and metastasis formation. The loss of KiSS1 by tumor cells has been associated with a metastatic phenotype but the mechanistic insights of this process are still unknown.

Experimental design: The blockade of the metastatic process at early stage is a hot topic in cancer research. We studied the role of KiSS1 on proliferation, invasiveness, migration abilities of mesothelioma cell lines focusing on the effect on epithelial-to-mesenchymal transition (EMT).

Results: Treatment with the KiSS1 peptide or with a synthesis peptide with longer half-life, the FTM080, significantly inhibited cell proliferation, migration and invasion of mesothelioma cell lines; the same treatment reduced the activity of MMP-2 and MMP-9 determining consequently a marked reduction in the invasiveness of primary tumors and metastases. Thespecificexpression of EMT markers, as E-caderin, Vimentin, Slug and Snail, suggested the inhibition of EMT after treatment with KiSS1 as well as the preservation of epithelial components.

Conclusion: Our results support anti-proliferative effect of KiSS1 in cancer cells and suggest that targeting the KiSS1/GPR54 system may represent a novel therapeutic approach for mesothelioma.

Keywords: EMT; KiSS1; biomarker; mesothelioma; metastasis.

Figure 1. Expression analysis of KiSS1/GPR54 system in mesothelioma cell lines

(A) qPCR and (B) Western blot analysis on total mRNA and protein of KiSS1 and GPR54 from mesothelioma cell lines: H2452, H28 and MSTO. Gene and protein expression levels was determined by normalizing to GAPDH and α-Tubulin, for qPCR and Western Blot respectively.

Figure 2. Effect of Kisspeptin and FTM080 on the proliferation and the invasiveness of mesothelioma cell lines

(A-B) Cell viability, (C-D) invasion ability. Data are the average ± SD of three independent experiments, each performed in triplicate. Asterisks indicate statistical significance, as determined by the Student-t test (^*** P ≤ 0.001).

Figure 3. Effect of Kisspeptin and FTM080 on the migration and colonies formation of mesothelioma cell lines

(A-B) Colony formation assay, (C) migration ability. Data are the average ± SD of three independent experiments, each performed in triplicate. Asterisks indicate statistical significance, as determined by the Student-t test (^** P ≤ 0.01; ^*** P ≤ 0.001).

Figure 4. Effect of Kisspeptin on intracellular signaling in mesothelioma cell lines: Epcam, Zymography, Western Blot

(A) Flow cytometry analysis with EpCAM staining in H28 and H2452 mesothelioma cell lines after treatment with Kp-10. Asterisks indicate statistical significance, as determined by the Student-t test (^** P ≤ 0.01). (B) MMP-2 and MMP-9 activities determined by gelatin zymography in the conditioned media of H28 and H2452 mesothelioma cell lines before and after treatment with Kp-10. (C) Western blot analysis for EMT-related protein E-cadherin, Vimentin, Snail, Slug and Survivin were performed on protein lysates from both H28 and H2452 mesothelioma cell lines following treatment with the indicate concentration of Kp-10. α-Tubulin was included as a loading control.

Figure 5. In vivo effects of the FTM080 treatment

Percentage of human Alu sequences in the lungs of athymic nude mice after tail vein injection with H2452 mesothelioma cells and the indicated treatments. Data are the average ± SD of three independent experiments, each performed in triplicate. Asterisks indicate statistical significance, as determined by the Student-t test (^*** P ≤ 0.001).