A Peptidic Thymidylate-Synthase Inhibitor Loaded on Pegylated Liposomes Enhances the Antitumour Effect of Chemotherapy Drugs in Human Ovarian Cancer Cells

Abstract

There is currently no effective long-term treatment for ovarian cancer (OC) resistant to poly-chemotherapy regimens based on platinum drugs. Preclinical and clinical studies have demonstrated a strong association between development of Pt-drug resistance and increased thymidylate synthase (hTS) expression, and the consequent cross-resistance to the hTS inhibitors 5-fluorouracil (5-FU) and raltitrexed (RTX). In the present work, we propose a new tool to combat drug resistance. We propose to treat OC cell lines, both Pt-sensitive and -resistant, with dual combinations of one of the four chemotherapeutic agents that are widely used in the clinic, and the new peptide, hTS inhibitor, [D-Gln⁴]LR. This binds hTS allosterically and, unlike classical inhibitors that bind at the catalytic pocket, causes cell growth inhibition without inducing hTS overexpression. The dual drug combinations showed schedule-dependent synergistic antiproliferative and apoptotic effects. We observed that the simultaneous treatment or 24h pre-treatment of OC cells with the peptide followed by either agent produced synergistic effects even in resistant cells. Similar synergistic or antagonistic effects were obtained by delivering the peptide into OC cells either by means of a commercial delivery system (SAINT-PhD) or by pH sensitive PEGylated liposomes. Relative to non-PEGylated liposomes, the latter had been previously characterized and found to allow macrophage escape, thus increasing their chance to reach the tumour tissue. The transition from the SAINT-PhD delivery system to the engineered liposomes represents an advancement towards a more drug-like delivery system and a further step towards the use of peptides for in vivo studies. Overall, the results suggest that the association of standard drugs, such as cDDP and/or 5-FU and/or RTX, with the novel peptidic TS inhibitor encapsulated into PEGylated pH-sensitive liposomes can represent a promising strategy for fighting resistance to cDDP and anti-hTS drugs.

Keywords: 5-fluorouracil; drug-resistance; human thymidylate synthase peptidic-inhibitors; ovarian cancer; pH-sensitive PEGylated liposomes; raltitrexed.

Figure 1

Effects of scheduled combinations of the [D-Gln4]LR peptide with cDDP, paclitaxel, 5FU and RTX on the SQ values in A2780 and A2780/CP cell lines. (A) concurrent combinations for 72 h. (B) Sequential combinations, as described in Section 4. The bars represent the mean of duplicate cell counts on three separate experiments and indicate the result of the inhibition of drug combination divided by the sum of the inhibition of a single drug to obtain the values of SQ. Error bars, SD.

Figure 2

Effects of scheduled combinations of [DGln4]LR peptide with cDDP, paclitaxel, 5FU and RTX on the SQ values in 2008 and C13* cell lines. (A) concurrent combinations for 72 h. (B) Sequential combinations as described in Section 4. The bars represent the mean of duplicate cell counts on three separate experiments and indicate the results of the inhibition of drug combinations divided by the sum of the inhibition of a single drug to obtain the values of SQ. Error bars, SD.

Figure 3

Effects of scheduled combinations of [DGln4]LR peptide with cDDP, paclitaxel, 5FU and RTX on the SQ values in IGROV-1 cell line. (Left panel) Concurrent combinations for 72 hr. (Right panel) Sequential combinations as described in Section 4. The bars represent the mean of duplicate cell counts on three separate experiments and indicate the results of the inhibition of drug combinations divided by the sum of the inhibition of a single drug to obtain the values of SQ. Error bars, SD.

Figure 4

Heatmap of the synergism quotient values of the tested combinations (rows) against the different cell lines (columns). Colour code: red, SQ > 1; blue, SQ < 1. The reported dendrogram was built based on the dissimilarity matrix using Euclidean distances and the complete linkage method. ld: lower dose; hd: higher dose; Pacli: paclitaxel, Seq I, II, III: combination sequences I, II and III as described in the text.

Figure 5

Effect of the [DGln4]LR peptide and 5-FU alone and in combination on the cell cycle phase distribution of 2008 and C13* cells by cytofluorimetric analysis of the DNA content by PI staining. After a 72 exposure to 5 µM [DGln4]LR and to the indicated concentrations of 5-FU alone or in concurrent combinations. Cells were processed according to methods described in Section 4. The inserted numbers indicate the percentages of cells in the different phases of the cell cycle and are the mean of two/three experiments. The error bars are omitted for a clearer visualization.

Figure 6

Effect of the [DGln4]LR peptide and cDDP alone and in combination on the cell cycle phase distribution of 2008 and C13* cells by cytofluorimetric analysis of the DNA content by PI staining. After a 72 exposure to 5 µM [DGln4]LR and 5 µM (2008 cells) or 10 µM (C13* cells) cDDP or 20 nM RTX alone and in concurrent combinations, cells were processed according to Section 4. The inserted numbers indicate the percentages of cells in the different phases of the cell cycle and are the mean of two/three experiments. The error bars were omitted for a clearer visualization.

Figure 7

Time course of the [D-Gln4]LR release percentage from [D-Gln4]LR-PEGylated liposomes in phosphate buffer saline (PBS; pH 7.4). Error bars, SD; where not visible, error bars did not exceed symbol size.

Figure 8

MTT test on C13*, 2008 and IGROV-1 cell lines. Cells were incubated with increasing amounts of [D-Gln4]LR-PpHL and PpHL (A) for 15 h, followed by a further 48-h incubation after the removal of the treatment. The results were expressed as percentage of cell growth with respect to the control (untreated cells), set at 100% of viability. Error bars, SD. Comparison between groups was performed by the ANOVA one-way test. Statistical significance levels were defined as * p < 0.05, ** p < 0.01 and *** p < 0.005. (B) The synergism of cell growth inhibition is reported as synergism quotient (SQ). The Concurrent chart corresponds to simultaneous liposomes + drug-exposure; D+L chart corresponds to sequential exposure in which the drugs (cDDP or RTX) was given 24 h before liposomes; L+D chart corresponds to the reversed sequential exposure. Error bars, SD.