Ru(II)-Fenamic-Based Complexes as Promising Human Ovarian Antitumor Agents: DNA Interaction, Cellular Uptake, and Three-Dimensional Spheroid Models

Abstract

Cancer resistance to chemotherapeutic agents such as cisplatin presents a significant challenge, leading to treatment failure and poor outcomes. Novel metal-based compounds offer a promising strategy to overcome drug resistance and to enhance efficacy. Four Ru(II) complexes with fenamic acid derivatives were synthesized and characterized: [Ru(L)(bipy)(dppp)]PF₆, where L represents fenamic acid (HFen, complex 1), mefenamic acid (HMFen, complex 2), tolfenamic acid (HTFen, complex 3), and flufenamic acid (HFFen, complex 4). Their composition was supported by molar conductivity, elemental analysis, Fourier transform infrared spectroscopy, ultraviolet-visible spectroscopy, mass spectrometry, and ³¹P{¹H}, ¹H, and ¹³C nuclear magnetic resonance, with the crystal structure of complex 1 confirmed via X-ray diffraction. Complexes 1-4 exhibited notable cytotoxicity against tested cell lines, particularly A2780 and A2780cisR (cisplatin-resistant ovarian tumors), compared to MDA-MB-231 (breast) and A549 (lung) lines. Mechanistic studies revealed weak DNA interactions through minor grooves or electrostatic binding. Cellular uptake assays showed effective internalization of complexes 1 (3.6%) and 2 (4.5%), correlating with potent IC₅₀ values. These complexes also altered cell morphology, reduced cell density, and inhibited colony formation in the A2780 cells. Staining assays indicated induced cell death and organelle damage, highlighting their potential as promising antitumor agents.

Scheme 1. Synthetic Procedures for Complexes 1–4

Figure 1

Crystallographic structure of complex 1 and the coordinated atoms and ruthenium labeled, with ellipsoids drawn at the 30% thermal probability level.

Figure 2

Interaction of complexes 1–4 with DNA. (A) Relative viscosity of CT-DNA after the addition of different concentrations of complexes 1–4 and thiazole orange at 25 °C. (B) Fluorescence emission spectra of DNA–Hoechst without the complex and with increasing amounts of complexes 1–4 (λ_exc = 343 nm).

Figure 3

Representation of microscopic images illustrating the changes in the cellular morphology of A2780 ovarian cancer cells, 0 and 48 h after treatment with compounds 1 and 2 at concentrations of 0.5 × IC₅₀, IC₅₀, and 2 × IC₅₀. The negative control was DMSO. The images were captured using a NIKON ECLIPSE TS100 microscope at 10× magnification.

Figure 4

Illustration of fluorescence microscopic images showing changes in the morphology of the A2780 ovarian cancer cells. Propidium iodide fluorescence after treatment with compounds 1 and 2 for 48 h at concentrations of 0.5 × IC₅₀, IC₅₀, and 2 × IC₅₀. The negative control used was DMSO. The images were captured using a CELENA microscope at 10× magnification.

Figure 5

Representation of changes in the morphology of A2780 ovarian cancer cells by fluorescence microscopic images with Green Plasma and DAPI markers. Data were obtained after treatment with compounds 1 and 2 for 48 h at concentrations of 0.5 × IC₅₀, IC₅₀, and 2 × IC₅₀. The negative control was DMSO. The images were captured using a CELENA microscope at 10× magnification.

Figure 6

Effect of complexes 1 and 2 on colony formation of A2780 tumor cells (A) Images of colonies at different concentrations of the compounds. The experiment was conducted in triplicate. (B) Illustration of the quantitative data that represent the colony number and colony size related to the concentrations of the complexes. The negative control was DMSO, and the data are presented as the mean ± standard deviation of three independent measurements. The statistical analysis was conducted by using a one-way analysis of variance test (**p < 0.01, ***p < 0.001, and ****p < 0.0001).

Figure 7

Percentage of ruthenium uptake in A2780 cells after incubation with complexes 1 and 2 at a concentration of 1 μM.

Figure 8

(A) Effect of complex 1, at different concentrations, on the cell morphology of spheroids of the A2780 tumor line, for 144 h. (B) Graph of spheroid diameter vs treatment time. (C) Fluorescence microscopy with DAPI and PI markers, after treatment for 144 h. The images were recorded using a CELENA microscope at 4x magnification. The negative control was DMSO.