We investigated the use of amphiphilic, protease-cleavable peptides as encapsulation moieties for hydrophobic metallodrugs, in order to enhance their bioavailability and consequent activity. Two hydrophobic, gold-containing anticancer agents varying in aromatic ligand distribution (Au(I)-N-heterocyclic carbene compounds 1 and 2) were investigated. These were encapsulated into amphiphilic decapeptides that form soluble filamentous structures with hydrophobic cores, varying supramolecular packing arrangements and surface charge. Peptide sequence strongly dictates the supramolecular packing within the aromatic core, which in turn dictates drug loading. Anionic peptide filaments can effectively load 1, and to a lesser extent 2, while their cationic counterparts could not, collectively demonstrating that loading efficiency is dictated by both aromatic and electrostatic (mis)matching between drug and peptide. Peptide nanofilaments were nontoxic to cancerous and noncancerous cells. By contrast, those loaded with 1 and 2 displayed enhanced cytotoxicity in comparison to 1 and 2 alone, when exposed to Caki-1 and MDA-MB-231 cancerous cell lines, while no cytotoxicity was observed in noncancerous lung fibroblasts, IMR-90. We propose that the enhanced in vitro activity results from the enhanced proteolytic activity in the vicinity of the cancer cells, thereby breaking the filaments into drug-bound peptide fragments that are taken up by these cells, resulting in enhanced cytotoxicity toward cancer cells.
This review focuses on studies of coordination and organometallic compounds as potential chemotherapeutics against triple negative breast cancer (TNBC) which has one of the poorest prognoses and worst survival rates from all breast cancer types. At present, chemotherapy is still the standard of care for TNBC since only one type of targeted therapy has been recently developed. References for metal-based compounds studied in TNBC cell lines will be listed, and those of metal-specific reviews, but a detailed overview will also be provided on compounds studied in vivo (mostly in mice models) and those compounds for which some preliminary mechanistic data was obtained (in TNBC cell lines and tumors) and/or for which bioactive ligands have been used. The main goal of this review is to highlight the most promising metalbased compounds with potential as chemotherapeutic agents in TNBC.
Supramolecular self‐assembly in biological systems holds promise to convert and amplify disease‐specific signals to physical or mechanical signals that can direct cell fate. However, it remains challenging to design physiologically stable self‐assembling systems that demonstrate tunable and predictable behavior. Here, the use of zwitterionic tetrapeptide modalities to direct nanoparticle assembly under physiological conditions is reported. The self‐assembly of gold nanoparticles can be activated by enzymatic unveiling of surface‐bound zwitterionic tetrapeptides through matrix metalloprotease‐9 (MMP‐9), which is overexpressed by cancer cells. This robust nanoparticle assembly is achieved by multivalent, self‐complementary interactions of the zwitterionic tetrapeptides. In cancer cells that overexpress MMP‐9, the nanoparticle assembly process occurs near the cell membrane and causes size‐induced selection of cellular uptake mechanism, resulting in diminished cell growth. The enzyme responsiveness, and therefore, indirectly, the uptake route of the system can be programmed by customizing the peptide sequence: a simple inversion of the two amino acids at the cleavage site completely inactivates the enzyme responsiveness, self‐assembly, and consequently changes the endocytic pathway. This robust self‐complementary, zwitterionic peptide design demonstrates the use of enzyme‐activated electrostatic side‐chain patterns as powerful and customizable peptide modalities to program nanoparticle self‐assembly and alter cellular response in biological context.
Dedicated to Prof. Adoración Gómez-Quiroga, cancer survivor and outstanding medicinal inorganic chemist.Triple negative breast cancer (TNBC) is one of the breast cancers with poorer prognosis and survival rates. TNBC has a disproportionally high incidence and mortality in women of African descent. We report on the evaluation of Ru-IM (1), a watersoluble organometallic ruthenium compound, in TNBC cell lines derived from patients of European (MDA-MB-231) and African (HCC-1806) ancestry (including IC 50 values, cellular and organelle uptake, cell death pathways, cell cycle, effects on migration, invasion, and angiogenesis, a preliminary proteomic analysis, and an NCI 60 cell-line panel screen). 1 was previously found highly efficacious in MDA-MB-231 cells and xenografts, with little systemic toxicity and preferential accumulation in the tumor. We observe a similar profile for this compound in the two cell lines studied, which includes high cytotoxicity, apoptotic behavior and potential antimetastatic and antiangiogenic properties. Cytokine M-CSF, involved in the PI3/AKT pathway, shows protein expression inhibition with exposure to 1. We also demonstrate a p53 independent mechanism of action.
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{(Ph3PN-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-Ph2PN-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.
Peptide materials are promising for various biomedical applications; however, a significant concern is their lack of stability and rapid degradation in vivo due to non-specific proteolysis. For materials specifically designed to respond to disease-specific proteases, it would be desirable to retain high susceptibility to target proteases while minimizing the impact of non-specific proteolysis. We describe N-terminal acetylation as a simple synthetic modification of amphiphilic self-assembling peptides that contain an MMP-9-cleavable segment and form soluble, nanoscale filaments. We found that the N-terminus capping of these peptides did not significantly impact their self-assembly behavior, critical aggregation concentration, or ability to encapsulate hydrophobic payloads. By contrast, their proteolytic stability in human plasma (especially for anionic peptide sequences) was considerably increased while susceptibility to hydrolysis by MMP-9 was retained when compared to non-acetylated peptides, especially during the first 12 h. We note, however, that due to the longer time scale required for in vitro studies (72 h), non-specific proteolysis of both anionic acetylated peptides leads to similar activity in vitro despite differing MMP-9 kinetics during the early stages. Overall, the enhanced stability against non-specific proteases, combined with the ability of these nanofilaments to enhance the effectiveness of gold-based drugs toward cancerous cells compared to healthy cells, brings these acetylated peptide filaments a step closer toward clinical translation.
Purpose: Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer that is defined by the absence of expression of progesterone receptor, estrogen receptor, and human epidermal growth factor receptor 2. TNBC has a higher incidence in younger women and those of African descent. Due to the inability to target a receptor, treatment is mostly limited to anthracycline and taxane-based nonspecific chemotherapy. Within the past two years more therapies have become available for later stage TNBC, such as antibody-drug conjugate sacituzumab govitecan-hziy. A potential water-soluble ruthenium-based chemotherapeutic agent was developed in our lab, showing high cytotoxicity against different cancer types in a National Cancer Institute 60-cell line panel. A preliminary in vivo study of this complex showed a pronounced 56% reduction of tumor growth with a TNBC MDA-MB-231 cell line xenograft mouse model, with little system toxicity and preferential accumulation of the complex in tumor tissue. These studies prompted us to further evaluate it in TNBC cell lines MDA-MB-231 and HCC-1806, derived from Caucasian and African ancestry patients respectively. This investigation revealed significant anti-angiogenic, anti-invasive, and anti-migratory properties for the complex, along with subcellular accumulation in the mitochondria and an apoptotic mechanism of cell death in both cell lines. Preliminary proteomic analysis was completed to understand the molecular pathway involved, which was determined to be P53-independent. Macrophage colony stimulating factor (M-CSF) showed an inhibition of protein expression, implicating the ruthenium complex as a potential inhibitor of phosphoinositide 3-kinases/protein kinase B (PI3K/Akt) pathway which acts downstream of M-CSF, and is involved with enhanced cancerous proliferation. In light of these findings, we report here on in vitro studies focused on further investigating the involvement of the PI3K/Akt molecular pathway and mechanism of action of this ruthenium complex. Experimental Design: Western blot studies directed toward key markers in the PI3K/Akt pathway will be assessed, which include Akt, c-Raf, PI3K, PTEN, and PDK1. Furthermore, mitochondrial involvement was determined through reactive oxygen species (ROS) generation and changes to mitochondrial membrane potential (MMP). Results: A significant increase of ROS generation was seen with exposure to the compound, along with slight MMP depolarization. Western blot analysis may further implicate an inhibition of the PI3K/Akt pathway. Conclusions: Our results show that the mechanistic action of the tested ruthenium complex has mitochondrial involvement, with inhibitory effects in the PI3K/Akt pathway. The very significant anti-cancer properties outlined in the preclinical evaluation of this complex will be further investigated in PDX mice models. Citation Format: Nazia Nayeem, Karen Hubbard, Maria Contel. Exploring the mode of action of a highly efficacious ruthenium-based chemotherapeutic agent in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1845.
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