Prevention of prostate cancer by natural product MDM2 inhibitor GS25: in vitro and in vivo activities and molecular mechanisms.

Prostate cancer remains a major health problem in the USA and worldwide. There is an urgent need to develop novel approaches to preventing primary and metastatic prostate cancer. We have identified 25-OCH3-protopanaxadiol (GS25), the most active ginsenoside that has been identified so far; it has potent activity against human cancers, including prostate cancer. However, it has not been proven if GS25 could be a safe and effective agent for cancer prevention. In this study, we used the TRAMP model and clearly demonstrated that GS25 inhibited prostate tumorigenesis and metastasis with minimal host toxicity. Mechanistically, GS25 directly bound to the RING domain of MDM2, disrupted MDM2-MDMX binding and induced MDM2 protein degradation, resulting in strong inhibition of prostate cancer cell growth and metastasis, independent of p53 and androgen receptor status. In conclusion, our in vitro and in vivo data support the potential use of GS25 in prevention of primary and metastatic prostate cancer.

Arsenite Targets the RING Finger Domain of Rbx1 E3 Ubiquitin Ligase to Inhibit Proteasome-Mediated Degradation of Nrf2.

Activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant response signaling pathway is a major mechanism for the cellular defense against oxidative stress. Arsenite, a widespread contaminant in drinking water, is known to induce oxidative stress and activate the Nrf2-dependent signaling pathway through the stabilization of the Nrf2 protein by inhibiting its ubiquitination via the Cul3-Rbx1-Keap1 (cullin 3, RING-box 1, and Kelch-like ECH-associated protein 1) E3 ubiquitin ligase, and its degradation by the 26S proteasome, though the underlying mechanism, remains elusive. In the present study, we demonstrated that arsenite could bind to the RING finger domain of Rbx1 in vitro and in cells, which led to the suppression of Cul3-Rbx1 E3 ubiquitin ligase activity, thereby impairing the Nrf2 ubiquitination and activating the Nrf2-induced antioxidant signaling pathway. Our finding provided novel insight into arsenic toxicity by uncovering a distinct mechanism accounting for arsenite-induced Nrf2 activation.

Biochemical and biophysical characterization of ruthenation of BRCA1 RING protein by RAPTA complexes and its E3 ubiquitin ligase activity.

RAPTA compounds, ([Ru(η6-arene)(PTA)Cl2], PTA = 1,3,5-triaza-7-phosphaadamantane), have been reported to overcome drug resistance in cisplatin resistant cells. However, the exact mechanism of these complexes is still largely unexplored. In this study, the interaction of some RAPTA compounds with the N-terminal fragment of the BRCA1 RING domain protein was investigated. The binding of the RAPTA compounds to the BRCA1 protein resulted in a release of Zn2+ ions in a dose and time dependent manner, as well as thermal alteration of ruthenated-BRCA1 proteins. Electron Transfer Dissociation (ETD) fragmentation mass spectrometry revealed the preferential binding sites of the RAPTA complexes on the BRCA1 zinc finger RING domain at a similar short peptide stretch, Cys24Lys25Phe26Cys27Met28Leu29 and Lys35 (residues 44-49 and 55 on full length BRCA1). Changes in the conformation and binding constants of ruthenium-BRCA1 adducts were established, resulting in inactivation of the RING heterodimer BRCA1/BARD1-mediated E3 ubiquitin ligase function. These findings could provide mechanistic insight into the mode of action of RAPTA complexes for on tested BRCA1 model protein.

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 656.29(2+) ion indicated that the ion arose from [Pt(NH3)2(OH)]+ bound to the BRCA1 peptide (111)ENNSPEHLK(119). 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 degrees , 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.