Abstract
Background: Peptide hormone-based targeted tumor therapy is an approved strategy to selectively block the tumor growth and spreading. The gonadotropin-releasing hormone receptors (GnRH-R) overexpressed on different tumors (e.g., melanoma) could be utilized for drug-targeting by application of a GnRH analog as a carrier to deliver a covalently linked chemotherapeutic drug directly to the tumor cells. In this study our aim was (i) to analyze the effects of GnRH-drug conjugates on melanoma cell proliferation, adhesion and migration, (ii) to study the mechanisms of tumor cell responses, and (iii) to compare the activities of conjugates with the free drug.Results: In the tested conjugates, daunorubicin (Dau) was coupled to 8Lys of GnRH-III (GnRH-III(Dau=Aoa)) or its derivatives modified with 4Lys acylated with short-chain fatty acids (acetyl group in [4Lys(Ac)]-GnRH-III(Dau=Aoa) and butyryl group in [4Lys(Bu)]-GnRH-III(Dau=Aoa)). The uptake of conjugates by A2058 melanoma model cells proved to be time dependent. Impedance-based proliferation measurements with xCELLigence SP system showed that all conjugates elicited irreversible tumor growth inhibitory effects mediated via a phosphoinositide 3-kinase-dependent signaling. GnRH-III(Dau=Aoa) and [4Lys(Ac)]-GnRH-III(Dau=Aoa) were shown to be blockers of the cell cycle in the G2/M phase, while [4Lys(Bu)]-GnRH-III(Dau=Aoa) rather induced apoptosis. In short-term, the melanoma cell adhesion was significantly increased by all the tested conjugates. The modification of the GnRH-III in position 4 was accompanied by an increased cellular uptake, higher cytotoxic and cell adhesion inducer activity. By studying the cell movement of A2058 cells with a holographic microscope, it was found that the migratory behavior of melanoma cells was increased by [4Lys(Ac)]-GnRH-III(Dau=Aoa), while the GnRH-III(Dau=Aoa) and [4Lys(Bu)]-GnRH-III(Dau=Aoa) decreased this activity.Conclusion: Internalization and cytotoxicity of the conjugates showed that GnRH-III peptides could guard Dau to melanoma cells and promote antitumor activity. [4Lys(Bu)]-GnRH-III(Dau=Aoa) possessing the butyryl side chain acting as a “second drug” proved to be the best candidate for targeted tumor therapy due to its cytotoxicity and immobilizing effect on tumor cell spreading. The applicability of impedimetry and holographic phase imaging for characterizing cancer cell behavior and effects of targeted chemotherapeutics with small structural differences (e.g., length of the side chain in 4Lys) was also clearly suggested.
Highlights
The application of more selective, targeted drugs has become increasingly important in the treatment of tumors, where the use of chemotherapeutics with low therapeutic index is restricted by the adverse events coming from the toxicity of these drugs to normal cells [1]
Internalization and cytotoxicity of the conjugates showed that GnRH-III peptides could guard Dau to melanoma cells and promote antitumor activity. [4Lys(Bu)]-GnRH-III(Dau=Aoa) possessing the butyryl side chain acting as a “second drug” proved to be the best candidate for targeted tumor therapy due to its cytotoxicity and immobilizing effect on tumor cell spreading
The chemotherapeutic drug targeting is often based on the receptors for certain peptide hormones that are preferentially expressed by cancer cells
Summary
The application of more selective, targeted drugs has become increasingly important in the treatment of tumors, where the use of chemotherapeutics with low therapeutic index is restricted by the adverse events coming from the toxicity of these drugs to normal cells [1]. It is highly advantageous that several native GnRH analogs, including the two human isoforms (GnRH-I and GnRH-II), and their synthetic derivatives have been reported to exert an antiproliferative effect in different types of tumors related (e.g., breast, endometrial cancer [5]) and unrelated (e.g., melanoma, colon carcinoma [6,7]) to reproductive organs Considering these aspects, the GnRH could serve as a targeting unit with the aim to increase the concentration of an attached cytotoxic drug at the tumor cells overexpressing GnRH-R, and to decrease the unnecessary exposure of normal cells lacking GnRH-R [8]. In this study our aim was (i) to analyze the effects of GnRH-drug conjugates on melanoma cell proliferation, adhesion and migration, (ii) to study the mechanisms of tumor cell responses, and (iii) to compare the activities of conjugates with the free drug
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