Comparative cell biological study of in vitro antitumor and antimetastatic activity on melanoma cells of GnRH-III-containing conjugates modified with short-chain fatty acids

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.

Enhanced In Vitro Antitumor Activity of GnRH-III-Daunorubicin Bioconjugates Influenced by Sequence Modification

“…All three cell lines display a band at approximately 38 kDa, which can be considered as the full-length human GnRH-R (Supporting Information S18) [47]. Apart from that, we detected additional bands with higher molecular weights (55–70 kDa), which is in accordance with reported findings and can be assumed to be glycosylated forms of GnRH-Rs [48,49,50].…”

Apoptotic Effects of Drug Targeting Conjugates Containing Different GnRH Analogs on Colon Carcinoma Cells

“…In our previous studies, GnRH-R expression was verified on our HT-29 model cells by Western blot analysis [22,42]. In these studies, the short-term antitumor activity of Dau–GnRH conjugates with 4 Ser as well as the enhanced cytostatic effect of the GnRH-III-based conjugate modified with 4 Lys(Bu) were also determined on HT-29 cells by alamarBlue-assay [21,42].…”

“…One of the most effective strategies to improve the antitumor activity and other biochemical properties of GnRH-III and its conjugate was to replace the Ser in position 4 with acylated Lys [13,21]. Based on the results of our systematic investigation of this kind of modification, GnRH-III-[ 4 Lys(Bu), 8 Lys(Dau=Aoa)]—containing Dau in position 8 and an apoptosis-inducing butyrate as a “second drug” on the side chain of 4 Lys—was proven to be the most potent conjugate due its enhanced antitumor activity, enzymatic stability and cellular uptake in different in vitro tumor models [21,22,23].…”

“…In the case of GnRH-I-[D-Lys 6 ] or GnRH-II-[D-Lys 6 ] with oxime-linked Dau, only a slight apoptotic activity could be determined on MCF-7 breast carcinoma cell line [12], while zoptarelin doxorubicin was proven to be more effective in inducing apoptosis in ovarian and endometrial carcinoma cell lines compared with the free drug [35]. Our recent results on melanoma cells indicated that in the case of the GnRH-III conjugate modified with butyrated 4 Lys GnRH-III[ 4 Lys(Bu), 8 Lys(Dau=Aoa)]), its antitumor effect was rather attributed to apoptotic activity, in contrast to the GnRH-III[ 8 Lys(Dau=Aoa)]-possessing 4 Ser, where the cell cycle blocking effect (arrest in the G2/M phase) was shown to be more prominent [22]. Although an increasing number of studies have focused on the determination of the apoptosis induced by different cytotoxic GnRH compounds, there are limited data on the underlying molecular mechanism.…”

“…Two sets of conjugates were synthesized by attaching Dau directly to the 6 D-Lys or 8 Lys (depending on the GnRH analog) via oxime linkage; one group with Ser in position 4 (GnRH-I-[ 4 Ser, 6 D-Lys(Dau=Aoa)], GnRH-II-[ 4 Ser, 6 D-Lys(Dau=Aoa)], GnRH-III[ 4 Ser, 8 Lys(Dau=Aoa)]), and a second group in which this Ser was replaced with butyrated Lys (GnRH-I-[ 4 Lys(Bu), 6 D-Lys(Dau=Aoa)], GnRH-II-[ 4 Lys(Bu), 6 D-Lys(Dau=Aoa)], GnRH-III[ 4 Lys(Bu), 8 Lys(Dau=Aoa)]) (Table 1). The idea behind examining these conjugates on colon carcinoma cells was that (i) GnRH-R is expressed on different colon cancer cells [39,40], (ii) different GnRH analog-containing conjugates could inhibit colorectal tumor growth both in vitro and in vivo [3,12,24,39,40,41], and (iii) modification with butyrated Lys has been already proven to be a successful strategy to improve the effectivity of conjugates in different types of GnRH-R-positive tumor cells (e.g., melanoma cells [22], colon and breast cancer cells [21,42,43]). In this paper, the synthesis and the analytical characterization of the above-mentioned GnRH conjugates with or without 4 Lys(Bu) were described, while their antiproliferative and apoptotic activity were studied by impedimetry (xCELLigence SP System), flow cytometry and quantitative real-time RT-PCR in HT-29 human colon carcinoma cell line.…”