Cellular responses of BRCA1-defective and triple-negative breast cancer cells and in vitro BRCA1 interactions induced by metallo-intercalator ruthenium(II) complexes containing chloro-substituted phenylazopyridine

Abstract

BackgroundTriple-negative breast cancer (TNBC) is defined by the absence of expression of estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2. Breast cancers with a BRCA1 mutation are also frequently triple-negative. Currently, there is a lack of effective therapies and known specific molecular targets for this aggressive breast cancer subtype. To address this concern, we have explored the cellular responses of BRCA1-defective and triple-negative breast cancer cells, and in vitro BRCA1 interactions induced by the ruthenium(II) complexes containing the bidentate ligand, 5-chloro-2-(phenylazo)pyridine.MethodsTriple-negative MDA-MB-231, BRCA1-defective HCC1937 and BRCA1-competent MCF-7 breast cancer cell lines were treated with ruthenium(II) complexes. The cytoxoxicity of ruthenium-induced breast cancer cells was evaluated by a real time cellular analyzer (RTCA). Cellular uptake of ruthenium complexes was determined by ICP-MS. Cell cycle progression and apoptosis were assessed using propidium iodide and Annexin V flow cytometry. The N-terminal BRCA1 RING protein was used for conformational and functional studies using circular dichroism and in vitro ubiquitination.ResultsHCC1937 cells were significantly more sensitive to the ruthenium complexes than the MDA-MB-231 and MCF-7 cells. Treatment demonstrated a higher degree of cytotoxicity than cisplatin against all three cell lines. Most ruthenium atoms were retained in the nuclear compartment, particularly in HCC1937 cells, after 24 h of incubation, and produced a significant block at the G2/M phase. An increased induction of apoptotic cells as well as an upregulation of p53 mRNA was observed in all tested breast cancer cells. It was of interest that BRCA1 mRNA and replication of BRCA1-defective cells were downregulated. Changes in the conformation and binding constants of ruthenium-BRCA1 adducts were observed, causing inactivation of the RING heterodimer BRCA1/BARD1-mediated E3 ubiquitin ligase activity.ConclusionsThis study has revealed the ability of ruthenium complexes to inhibit cell proliferation, induce cell cycle progression and apoptosis. Ruthenium treatment upregulated the marker genes involved in apoptosis and cell cycle progression while it downregulated BRCA1 mRNA and replication of HCC1937 cells. Our results could provide an alternative approach to finding effective therapeutic ruthenium-based agents with promising anticancer activity, and demonstrated that the BRCA1 RING domain protein was a promising therapeutic target for breast cancers.

Rationally Designed Ruthenium Complexes for Breast Cancer Therapy

“…It has been reported that ruthenium-based agents can be promising anticancer candidates to treat BRCA1-mutant breast cancers, frequently associated with TNBC [121,122]. The BRCA1 gene responds to DNA damage by being involved in cellular pathways for DNA repair, mRNA transcription, cell cycle regulation, and protein ubiquitination [123,124].…”

“…The role of BRCA1 is also to regulate chemotherapy-induced DNA damage [124]. Ratanaphan et al (2014) reported ruthenium(II) complexes that showed a promising cytotoxicity in a BRCA1 defective TNBC cell line, HCC1937 [121]. Importantly, complex (25) (Figure 10) induced significantly more cytotoxicity in BRCA1 defective-HCC1937 cells (IC 50 = 1.8 ± 0.1 µM) than in the BRCA1 wild-type cell lines MDA-MB-231 (IC 50 = 13.2 ± 0.3 µM) and MCF7 (IC 50 = 8.2 ± 0.1 µM), suggesting that the higher sensitivity of the BRCA1 defective breast cancer cells to (25) might be due to the inability of the dysfunctional BRCA1 to repair ruthenium-induced DNA damage.…”

The trans -[Ru(PPh 3 ) 2 ( N , N -dimethyl- N ′-thiophenylthioureato-k 2 O,S)(bipy)]PF 6 complex has pro-apoptotic effects on triple negative breast cancer cells and presents low toxicity in vivo

“…However, caspase-3 protein levels did not alter, while its gene expression is increased in all concentrations tested, probably due to shorter incubation times used in the gene expression assays compared to Western blotting assays. [55], which is commonly mutated in TNBC cells. These data suggest that ruthenium complexes can be an alternative to treat triple negative cancers [56].…”

Non-Platinum Metal Complexes as Potential Anti-Triple Negative Breast Cancer Agents

“…For example, Biancalana et al [57] observed that ruthenium complexes have good cytotoxic activity, with IC 50 values substantially lower than the values obtained with cisplatin on MDA-MB-231 cells. These differences could be due to the fact that they have different mechanisms of action; for example, the activation of p53-dependent or p53-independent checkpoints by cisplatin or ruthenium complexes, respectively [9,52,54,68]. Since platinum-based drugs and NPMBC act on different pathways, NPMBC could be a new therapeutic option for patients with TNBC resistant to platinum-based drugs.…”

“…However, both cisplatin chemotherapies and its analogs have been shown to have major drawbacks (i.e., intrinsic and acquired chemoresistance, high general toxicity and limited spectrum of activity) [26] which have motivated extensive investigations into alternative metal-based cancer therapies that effectively target both cancer cell proliferation and metastasis. In this sense, we can mention chemotherapy with gold compounds [27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45][46]-because they are nontoxic, nonimmunogenic and have good photothermal and optical properties, biocompatibility and stability [40,47,48]-and also with copper [49][50][51], ruthenium [16,[52][53][54][55][56][57][58][59], iron [56,[60][61][62][63][64][65], palladium [50], silver [66,…”