Adisorn Ratanaphan

In vitro ruthenation of human breast cancer suppressor gene 1 (BRCA1) by the antimetastasis compound RAPTA-C and its analogue CarboRAPTA-C.

The interaction of two ruthenium–arene–1,3,5‐triaza‐7‐phosphaadamantane compounds ([Ru(η6‐p‐cymene)Cl2(pta)] and [Ru(η6‐p‐cymene)(C6H6O4)(pta)], termed RAPTA‐C (3) and carboRAPTA‐C (4), resp.) with the DNA sequence of the human breast‐cancer suppressor gene 1 (BRCA1) has been studied using a range of techniques that probe conformation, cross‐linking, base specificity, restriction analysis, and in vitro inhibition of DNA polymerization. The study demonstrates that substitution of the two labile chloride ligands in 3 by the more stable cyclobutane‐1,1‐dicarboxylate ligand onto the RAPTA framework reduces the rate of reaction with DNA in a similar manner to the analogous Pt‐based drug pair cisplatin (1) and carboplatin (2), suggesting that hydrolysis may be a prerequisite to DNA binding with the Ru compounds. Moreover, the rate of DNA interaction for 3 is in a similar range to that of 2, despite the fact that these compounds have a different therapeutic profile. The similar rates of reaction contrasting with the different modes of activity suggests that the RAPTA compounds may be clinically useful against cancer cells that have developed resistance to Pt‐based therapies, particularly involving excision–repair mechanisms.

Cisplatin affects the conformation of apo form, not holo form, of BRCA1 RING finger domain and confers thermal stability.

The breast cancer suppressor protein 1 (BRCA1) has been shown to participate in genomic integrity maintenance. Preclinical and clinical studies have recently revealed that the inactivation of BRCA1 in cancer cells leads to chemosensitivity. Approaching the BRCA1 RING protein as a potentially molecular target for a platinum‐based drug might be of interest in cancer therapy. In the present study, the in vitro platination of the BRCA1 RING protein by the anticancer drug cisplatin was observed. The protein contained a preformed structure in the apo form with structural changes and resistance to limited proteolysis after Zn2+ binding. SDS‐PAGE and mass‐spectrometric analyses revealed that cisplatin preferentially formed monofunctional and bifunctional BRCA1 adducts. Tandem mass spectrometry (MS/MS) of the 656.292+ ion indicated that the ion arose from [Pt(NH3)2(OH)]+ bound to the BRCA1 peptide 111ENNSPEHLK119. The product‐ion spectrum revealed the Pt‐binding site on His117. Circular dichroism showed that the apo form, not holo form, of BRCA1 underwent more folded structural rearrangement upon cisplatin binding. Cisplatin‐bound protein exhibited an enhanced thermostability by 13°, resulting from the favorably intermolecular cross‐links driven by the free energy. Our findings demonstrated the first conformational and thermal evidences for a direct binding of cisplatin to the BRCA1 RING domain and could raise a possibility of selectively targeted treatment of cancer with less toxicity or improved response to conventional regimens.

Cisplatin-damaged BRCA1 exhibits altered thermostability and transcriptional transactivation

BRCA1 is a tumor suppressor gene. Its translated product has an important function in transcriptional activation and DNA repair pathways. Damaged BRCA1 due to cisplatin treatment may lead to loss of such functions. To address a potential drug target of BRCA1 for cisplatin treatment, we investigated the biophysical characterization and functional consequences of the 3/-terminal region of human BRCA1 after in vitro platination with cisplatin. To analyze the base/sequence specificity of cisplatin damage, the measurement for sensitivity of cisplatin-treated BRCA1 to restriction enzymes (EcoO109I and PvuII) and sequence gel analysis was conducted. The results suggested that the platination favorably occurred at the d(GpG) and the d(GpC) sites. An increase in drug concentrations resulted in increased interstrand crosslinks at the d(GpC) site. Cisplatin affected the transition temperature of the BRCA1 gene fragment in a biphasic fashion. DSC thermogram of DNA adducts was shifted to a lower transition temperature at lower cisplatin concentration. However, at higher drug concentration, the thermogram peaked at a slightly higher transition temperature with predominantly increased heat specific capacity. Reduction in cellular DNA repair of cisplatin-damaged plasmid DNA, using host cell reactivation assay, was a consequence of an increase in platination levels on the reporter gene. The GAL4-fused BRCA1 slightly enhanced the functional consequences of cisplatin-BRCA1 adducts 3 transcription of the reporter gene in the absence of GAL4 binding site. The transcriptional transactivation activity of cisplatin-modified BRCA1, when tested in “one-hybrid GAL4 transcriptional assay”, was inversely proportional to cisplatin doses. Furthermore, the transcriptional transactivation activity was dramatically diminished in the presence of a second expression vector containing multiple cisplatin-damaged sites. The data provide the first evidence for direct interaction of cisplatin with BRCA1 and raise the possibility of BRCA1 as a therapeutic target for platinum drug- based chemotherapy.

Altered DNA Binding and Amplification of Human Breast Cancer Suppressor Gene BRCA1 Induced by a Novel Antitumor Compound, [Ru(η6-p-phenylethacrynate)Cl2(pta)]

The ruthenium-based complex [Ru(η6-p-phenylethacrynate)Cl2(pta)] (pta = 1,3,5-triaza-7-phosphatricyclo-[3.3.1.1]decane), termed ethaRAPTA, is an interesting antitumor compound. The elucidation of the molecular mechanism of drug activity is central to the drug development program. To this end, we have characterized the ethaRAPTA interaction with DNA, including probing the sequence specific modified DNA structural stability and DNA amplification using the breast cancer suppressor gene 1 (BRCA1) of human breast and colon adenocarcinoma cell lines as models. The preference of ethaRAPTA base binding is in the order A > G > T > C. Once modified, the ethaRAPTA-induced BRCA1 structure has higher thermal stability than the modified equivalents of its related compound, RAPTA-C. EthaRAPTA exhibits a higher efficiency than RAPTA-C in inhibiting BRCA1 amplification. With respect to both compounds, the inhibition of BRCA1 amplification is more effective in an isolated system than in cell lines. These data provide evidence that will help to understand the process of elucidating the pathways involved in the response induced by ethaRAPTA.

BRCA1-Associated Triple-Negative Breast Cancer and Potential Treatment for Ruthenium-Based Compounds

Triple-negative breast cancer (TNBC) is defined by the absence of expression of estrogen receptor (ER), progesterone receptor (PR), and a lack of overexpression or amplification of human epidermal growth factor receptor 2 (HER2). The clinicopathological characteristics of TNBC include a high grading, a high rate of cell proliferation and a greater degree of chromosomal rearrangement. Patients with triple-negative breast cancer are more likely to be drug resistant and more difficult to treat, and are also frequently BRCA1 mutants. Methylation of the BRCA1 promoter region is associated with a reduction of the BRCA1 mRNA level. TNBC patients with a methylated BRCA1 had a better disease-free survival compared with those with non-methylated BRCA1. From a therapeutic perspective, the expression level of BRCA1 has been a major determinant of the responses to different classes of chemotherapy. BRCA1-dysfunctional tumors are hypersensitive to DNA damaging chemotherapeutic agents like platinum drugs. Although platinum based drugs are currently widely used as conventional chemotherapeutic drugs in breast cancer chemotherapy, their use has several disadvantages. It is therefore of interest to seek out alternative therapeutic metal-based compounds that could overcome the limitations of these platinum based drugs. Ruthenium-based compounds could be the most promising alternative to the platinum drugs. This review highlights the use of BRCA1 as a predictive marker as well as for a potential drug target for anticancer ruthenium compounds.

Substitution of aspartic acid with glutamic acid at position 67 of the BRCA1 RING domain retains ubiquitin ligase activity and zinc(II) binding with a reduced transition temperature

Breast cancer susceptibility protein 1 (BRCA1) participates in genomic integrity maintenance through DNA repair, cell cycle checkpoint, protein ubiquitination, and transcriptional regulation. The N-terminus of BRCA1 contains a RING domain which forms two Zn2+ binding sites in an interleaved fashion. A number of deleterious BRCA1 missense mutations, which predispose an individual to a subset of hereditary breast and ovarian cancers, have been identified in the RING domain. Disruption of Zn2+ binding sites and protein structure results in the inactivation of BRCA1 tumor suppression function. An unprecedented D67E BRCA1 mutation, identified in Thai familial breast cancer patients, is located in the vicinity of Zn2+ binding site II, and its pathogenic significance remains elusive. The present study revealed that the D67E BRCA1 RING protein assumes a preformed structure in the absence of Zn2+. The Zn2+-bound mutant protein was more folded, resulting in enhanced proteolytic resistance and dimerization. This indicated that the mutation retained Zn2+ binding, and barely perturbed the native global structure of the BRCA1 RING domain. The complex between D67E BRCA1 and BARD1 RING domains exhibited a substantial ubiquitin ligase activity compared with a defective complex containing the C61G BRCA1 mutation. However, the D67E mutation was slightly less stable toward thermal denaturation. This implies that the D67E mutation might be a neutral or mild cancer-risk modifier of other defective mechanisms underlying BRCA1-mutation-related breast cancer.

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.

A DNA Repair BRCA1 Estrogen Receptor and Targeted Therapy in Breast Cancer

BRCA1 is a key mediator of DNA repair pathways and participates in the maintenance of the genomic integrity of cells. The control of DNA damage repair mechanisms by BRCA1 is of great interest since molecular defects in this pathway may reflect a predictive value in terms of a cell’s sensitivity to DNA damaging agents or anticancer drugs. BRCA1 has been found to exhibit a hormone-dependent pattern of expression in breast cells. Wild-type BRCA1 is required for the inhibition of the growth of breast tumor cells in response to the pure steroidal ERα antagonist fulvestrant. Also a loss of BRCA1-mediated transcriptional activation of ERα expression results in increased resistance to ERα antagonists. Platinum-based drugs, poly(ADP-ribose) polymerase (PARP) inhibitors, and their combination are currently included in chemotherapy regimens for breast cancer. Preclinical and clinical studies in a BRCA1-defective setting have recently indicated a rationale for the use of these compounds against hereditary breast cancers. Initial findings indicate that neoadjuvant use of cisplatin results in high rates of complete pathological response in patients with breast cancer who have BRCA1 mutations. Cisplatin produces a better response in triple-negative breast cancer (TNBC) than in non-TNBC diseases in both the neoadjuvant and adjuvant settings. This implies that TNBC cells may harbor a dysfunctional BRCA1 repair pathway.

In Vitro Enhanced Sensitivity to Cisplatin in D67Y BRCA1 RING Domain Protein

BRCA1 is a tumor suppressor protein involved in maintaining genomic integrity through multiple functions in DNA damage repair, transcriptional regulation, cell cycle checkpoint, and protein ubiquitination. The BRCA1-BARD1 RING complex has an E3 ubiquitin ligase function that plays essential roles in response to DNA damage repair. BRCA1-associated cancers have been shown to confer a hypersensitivity to chemotherapeutic agents. Here, we have studied the functional consequence of the in vitro E3 ubiquitin ligase activity and cisplatin sensitivity of the missense mutation D67Y BRCA1 RING domain. The D67Y BRCA1 RING domain protein exhibited the reduced ubiquitination function, and was more susceptible to the drug than the D67E or wild-type BRCA1 RING domain protein. This evidence emphasized the potential of using the BRCA1 dysfunction as an important determinant of chemotherapy responses in breast cancer.

Host Cell Reactivation and Transcriptional Activation of Carboplatin-Modified BRCA1

The breast cancer susceptibility gene 1 (BRCA1) has been shown to maintain genomic stability through multiple functions in the regulation of DNA damage repair and transcription. Its translated BRCT (BRCA1 C-terminal domain) acts as a strong transcriptional activator. BRCA1 damaged by carboplatin treatment may lead to a loss of such functions. To address the possibility of the BRCA1 gene as a therapeutic target for carboplatin, we investigated the functional consequences of the 3′-terminal region of human BRCA1 following in vitro platination with carboplatin. A reduction in cellular BRCA1 repair of carboplatin-treated plasmid DNA, using a host cell reactivation assay, was dependent on the platination levels on the reporter gene. The transcriptional transactivation activity of the drug-modified BRCA1, assessed using a one-hybrid GAL4 transcriptional assay, was inversely proportional to the carboplatin doses. The data emphasized the potential of the BRCA1 gene to be a target for carboplatin treatment.