Anticancer activity of RAPTA-EA1 in triple-negative BRCA1 proficient breast cancer cells: single and combined treatment with the PARP inhibitor olaparib.

RAPTA-EA1 is a promising glutathione transferase (GSTP-1) inhibitor that has previously been shown to inhibit the growth of various breast cancer cells. We studied the anticancer activity of RAPTA-EA1 on triple-negative BRCA1 competent breast cancer MDA-MB-231 cells. MDA-MB-231 cells are significantly more sensitive to RAPTA-EA1 than MCF-7 cells. Treatment reveals a higher degree of cytotoxicity than cisplatin against both cell lines. Ruthenium accumulation in MDA-MB-231 cells is mainly in the nuclear fraction (43%), followed by the cytoplasm (30%), and the mitochondria (27%). RAPTA-EA1 blocks cell growth at the G2/M phase, leading to nuclear condensation and cell death. The compound slightly inhibits DNA replication of the 3,426-bp fragment of the BRCA1 exon 11 of the cells, with approximately 0.6 lesion per the BRCA1 fragment. The expression of BRCA1 mRNA and its protein in the Ru-treated cells is curtailed by 50-80% compared to the untreated controls. Growth inhibition of the triple-negative BRCA1 wild-type MDA-MB-231 and the sporadic BRCA1 wild-type MCF-7 cells by olaparib (a poly [ADP-ribose] polymerase (PARP) inhibitor) is dose-dependent, with MDA-MB-231 cells being two-fold less susceptible to the drug than MCF-7 cells. Combining olaparib with RAPTA-EA1 results in a combination index (CI) of 0.78 (almost additive) in MDA-MB-231 cells and 0.24 (potent synergy) in the MCF-7 cells. The PARP inhibitor alone differently regulates the expression of BRCA1 mRNA in both cell lines, whereas the olaparib-RAPTA-EA1 combination induces overexpression of BRCA1 mRNA in these cells. However, the expression level of the BRCA1 protein is dramatically reduced after treatment with the combined inhibitors, compared with the untreated controls. This observation highlights the cellular responses of triple-negative BRCA1 proficient breast cancer MDA-MB-231 cells to RAPTA-EA1 through BRCA1 inhibition and provides insights into alternative treatments for breast cancer.

Cellular responses of BRCA1-defective HCC1937 breast cancer cells induced by the antimetastasis ruthenium(II) arene compound RAPTA-T.

An organometallic ruthenium(II) arene compound, Ru(η6-toluene)(PTA)Cl2 (PTA = 1,3,5-triaza-7-phosphaadamantane), termed RAPTA-T, exerts promising antimetastatic properties. In this study, the effects of RAPTA-T on BRCA1-defective HCC1937 breast cancer cells have been investigated, and compared to its effects on BRCA1-competent MCF-7 breast cancer cells. RAPTA-T showed a very low cytotoxicity against both tested cells. Ruthenium is found mostly in the cytoplasmic compartment of both cells. Flow cytometric analysis reveals that the compound arrests the growth of both cells by triggering the G2/M phase that led to the induction of apoptosis. At equimolar concentrations, RAPTA-T causes much more cellular BRCA1 damage in HCC1937 than in MCF-7 cells, suppressing the expression of BRCA1 mRNA in both cell lines with the subsequent down-regulation of the BRCA1 protein. Interestingly, RAPTA-T exhibits an approximately fivefold greater ability to suppress the expression of the BRCA1 protein in HCC1937 than in MCF-7 cells. These data provide insights into the molecular mechanisms by which RAPTA-T exerts its effects on BRCA1-associated breast cancer cells.

Differential Cytotoxicity, Cellular Uptake, Apoptosis and Inhibition of BRCA1 Expression of BRCA1-Defective and Sporadic Breast Cancer Cells Induced by an Anticancer Ruthenium(II)-Arene Compound, RAPTA-EA1.

BACKGROUND: The RAPTA-EA1 complex [ruthenium(II)-arene 1,3,5-triaza-7-phosphaadamantane (pta) complex with an arene-tethered ethacrynic acid ligand] has been reported to overcome drug resistance that developed due to the current use of platinum-based treatments. However, the exact mechanism of action of RAPTA-EA1 remains largely unexplored and unknown. OBJECTIVE: Here we have further studied the effect of RAPTA-EA1 on BRCA1-defective HCC1937 breast cancer cells and compared its effects on BRCA1-competent MCF-7 breast cancer cells. METHOD: HCC1937 and MCF-7 breast cancer cells were treated with the RAPTA-EA1 complex. The cytotoxicity of ruthenium-induced cells was evaluated by a MTT assay. Cellular uptake of ruthenium was determined by ICP-MS. Cell cycle and apoptosis were assessed using a flow cytometer. Expression of BRCA1 mRNA and its encoded protein was quantitated by a real-time RT-PCR and Western blotting. RESULTS: Differences in cytotoxicity were correlated with the differential accumulations of ruthenium and the induction of apoptosis. The ruthenium complex caused dramatically more damage to the BRCA1 gene in the BRCA1-defective HCC1937 cells than to the BRCA1-competent MCF-7 cells. It decreased the expression of BRCA1 mRNA in the BRCA1-competent cells, while in contrast, its expression increased in the BRCA1-defective cells. However, the expression of the BRCA1 protein was significantly reduced in both types of breast cancer cells. CONCLUSION: The results presented here have demonstrated a differential cellular response for the BRCA1-defective and BRCA1-competent breast cancer cells to RAPTA-EA1. These findings have provided more insight into the actions and development of the ruthenium-based compounds for use for the treatment of breast cancer.

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.

BACKGROUND: Triple-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. METHODS: Triple-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. RESULTS: HCC1937 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. CONCLUSIONS: This 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.