Intracellular Localization Studies of the Luminescent Analogue of an Anticancer Ruthenium Iminophosphorane with High Efficacy in a Triple-Negative Breast Cancer Mouse Model

Abstract

The potential of ruthenium­(II) compounds as an alternative to platinum-based clinical anticancer agents has been unveiled after extensive research for over 2 decades. As opposed to cisplatin, ruthenium­(II) compounds have distinct mechanisms of action that do not rely solely on interactions with DNA. In a previous report from our group, we described the synthesis, characterization, and biological evaluation of a cationic, water-soluble, organometallic ruthenium­(II) iminophosphorane (IM) complex of p-cymene, ([(η6-p-cymene)­Ru­{(Ph3PN-CO-2N-C5H4)-κ-N,O}­Cl]­Cl (1 or Ru-IM), that was found to be highly cytotoxic against a panel of cell lines resistant to cisplatin, including triple-negative breast cancer (TNBC) MDA-MB-231, through canonical or caspase-dependent apoptosis. Studies on a MDA-MB-231 xenograft mice model (after 28 days of treatment) afforded an excellent tumor reduction of 56%, with almost negligible systemic toxicity, and a favored ruthenium tumor accumulation compared to other organs. 1 is known to only interact weakly with DNA, but its intracellular distribution and ultimate targets remain unknown. To gain insight on potential mechanisms for this highly efficacious ruthenium compound, we have developed two luminescent analogues containing the BOPIPY fluorophore (or a modification) in the IM scaffold with the general structure of [(η6-p-cymene)­Ru­{(BODIPY-Ph2PN-CO-2-NC5H4)-κ-N,O}­Cl]­Cl {BODIPY-Ph2P = 8-[(4-diphenylphosphino)­phenyl]-4,4-dimethyl-1,3,5,7-tetramethyl-2,6-diethyl-4-bora-3a,4a-diaza-s-indacene (3a) and 4,4-difluoro-8-[4-[[2-[4-(diphenylphosphino)­benzamido]­ethyl]­carbamoyl]­phenyl]-1,3,5,7-tetramethyl,2,6-diethyl-4-bora-3a,4a-diaza-s-indacene (3b)}. We report on the synthesis, characterization, lipophilicity, stability, luminescence properties, and cell viability studies in the TNBC cell line MDA-MB-231, nonmalignant breast cells (MCF10a), and lung fibroblasts (IMR-90) of the new compounds. The ruthenium derivative 3b was studied by fluorescence confocal microscopy. These studies point to a preferential accumulation of the compound in the endoplasmic reticulum, mitochondria, and lysosomes. Inductively coupled plasma optical emission spectrometry (ICP-OES) analysis also confirms a greater ruthenium accumulation in the cytoplasmic fraction, including endoplasmic reticulum and lysosomes, and a smaller percentage of accumulation in mitochondria and the nucleus. ICP-OES analysis of the parent compound 1 indicates that it accumulates preferentially in the mitochondria and cytoplasm. Subsequent experiments in 1-treated MDA-MB-231 cells demonstrate significant reactive oxygen species generation.

BODIPY as a Multifunctional Theranostic Reagent in Biomedicine: Self‐Assembly, Properties, and Applications

“…In addition, BODIPY can participate in the formation of self‐assembled systems such as metal‐based compounds for disease treatment and intracellular localization studies of analytes, which will shed light on the development of novel multifunctional theranostic agents. Based on their previous report of the organometallic ruthenium(II) iminophosphorane complex of p ‐cymene, [ 323 ] Contel and co‐workers [ 324 ] developed two luminescent analogs containing BODIPY moiety in the iminophosphorane scaffold. Fluorescent BODIPY moiety allows the distribution of these ruthenium complexes within biological tissues to be visualized.…”

LVVO-Ethyl Maltol-Based Metallodrugs (L2– = Tridentate ONO Ligands): Hydrophobicity, Hydrolytic Stability, and Cytotoxicity via ROS-Mediated Apoptosis

“…The live-cell confocal images were taken using blue channels (λ ex = 405 nm, λ em = 420−470 nm) for complexes and a red channel (λ ex = 635 nm, λ em = 650−740 nm) for MTDR. 86 The microscope was operated with Leica LAS-X software. The colocalization analyses were performed with ImageJ software.…”

Photoactive and Luminescent Transition Metal Complexes as Anticancer Agents: A Guiding Light in the Search for New and Improved Cancer Treatments

“…Miachin et al developed two luminescent derivatives of their triple-negative breast-canceractive ruthenium(II) piano stool complex; they reported that the addition of a fluorophore changed the biological characteristics of the resulting complexes (Figure 6: RU7). They found that the cytotoxicity was reduced from 2.72 ± 0.11 to 68.23 ± 8.2 in MDA-MB-231 cells, and ICPMS experiments found that the complexes also varied in their cell localization and uptake [58]. Although these findings justify the conclusion that adding a fluorophore can still be used to build a more complete picture of cellular accumulation and mechanism as reported, it is clear that developing a cytotoxic agent with inherent fluorescence would be more beneficial.…”