Targeting TGF-β-Mediated SMAD Signaling Pathway via Novel Recombinant Cytotoxin II: A Potent Protein from Naja naja oxiana Venom in Melanoma.

Afshin Derakhshani,Oronzo Brunetti,Parviz Vahedi,Morteza Shahmirzaie,Zahra Asadzadeh,Niloufar Sadat Nourbakhsh,Nima Hemmat,Nicola Silvestris,Behzad Baradaran,Leila Mobasheri,Mahdi Abdoli Shadbad,Hossein Safarpour

doi:10.3390/molecules25215148

Abstract

Since the current treatments have not resulted in the desired outcomes for melanoma patients, there is a need to identify more effective medications. Together with other snake venom proteins, cytotoxin-II has shown promising results in tumoral cells. In this study, recombinant cytotoxin-II (rCTII) was expressed in SHuffle® T7 Express cells, while the epitope mapping of rCTII was performed to reveal the antibody-binding regions of rCTII. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to assess the viability of SK-MEL-3 and HFF-2 cells after treating these cells with rCTII. The qRT-PCR was performed to evaluate the expression levels of matrix metallopeptidase 3 (MMP-3), SMAD2, SMAD3, caspase-8, caspase-9, and miR-214 in order to reveal the rCTII-induced signaling pathways in melanoma. Our results have shown that two regions of amino acids, 6–16 and 19–44, as predicted epitopes of this toxin, are essential for understanding the toxicity of rCTII. Treating the melanoma cells with rCTII substantially inhibited the transforming growth factor-beta (TGF-β)–SMAD signaling pathway and down-regulated the expression of MMP-3 and miR-214 as well. This cytotoxin also restored apoptosis mainly via the intrinsic pathway. The down-regulation of MMP-3 and miR-214 might be associated with the anti-metastatic property of rCTII in melanoma. The inhibitory effect of rCTII on the TGF-β signaling pathway might be associated with increased apoptosis and decreased cancer cell proliferation. It is interesting to see that the IC50 value of rCTII has been lower in the melanoma cells than non-tumoral cells, which may indicate its potential effects as a drug. In conclusion, rCTII, as a novel medication, might serve as a potent and efficient anticancer drug in melanoma.

Highlights

Due to its significant incidence and mortality, melanoma has remained one of the troublesome malignancies, especially among the fair-skinned populations [1]
Our result demonstrated that for the recombinant cytotoxin-II (rCTII) protein, the SMAD2 and SMAD3 were evaluated in the treated and untreated cell line to investigate the rCTII effect on the transforming expression levels were evaluated in the treated and untreated cell line to investigate the rCTII effect growth factor-beta (TGF-β) signaling pathway
Dysregulated apoptosis can endow the malignant cells with a selective survival superiority over non-tumoral cells [23]. This current study has revealed that rCTII can remarkably induce apoptosis, mostly via the intrinsic pathway, in melanoma cells

Summary

Introduction

Due to its significant incidence and mortality, melanoma has remained one of the troublesome malignancies, especially among the fair-skinned populations [1]. As a matter of fact, melanoma is responsible for most skin cancer-associated mortalities [2]. The available therapies have not resulted in the desired outcomes in affected patients [3,4]. Multiple animal-derived secretions, e.g., snake venom toxins, have shown promising results in tumoral cells [5,6]. The cytotoxicity of snake venoms has been associated with stimulating apoptosis and inhibiting the migration of tumoral cells [7,8]. Cytotoxin I and cytotoxin II are two cytotoxins from

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Discussion

Conclusion