A new gold(I) complex-Au(PPh3)PT is a deubiquitinase inhibitor and inhibits tumor growth.

BACKGROUND: Ubiquitin-proteasome system (UPS) is integral to cell survival by maintaining protein homeostasis, and its dysfunction has been linked to cancer and several other human diseases. Through counteracting ubiquitination, deubiquitinases (DUBs) can either positively or negatively regulate UPS function, thereby representing attractive targets of cancer therapies. Previous studies have shown that metal complexes can inhibit tumor growth through targeting the UPS; however, novel metal complexes with higher specificity for cancer therapy are still lacking. METHODS: We synthesized a new gold(I) complex, Au(PPh3)PT. The inhibitory activity of Au(PPh3)PT on the UPS and the growth of multiple cancer cell types were tested in vitro, ex vivo, and in vivo. Furthermore, we compared the efficacy of Au(PPh3)PT with other metal compounds in inhibition of UPS function and tumor growth. FINDINGS: Here we report that (i) a new gold(I) complex-pyrithione, i.e., Au(PPh3)PT, induced apoptosis in two lung cancer cell lines A549 and NCI-H1299; (ii) Au(PPh3)PT severely impaired UPS proteolytic function; (iii) Au(PPh3)PT selectively inhibited 19S proteasome-associated DUBs (UCHL5 and USP14) and other non-proteasomal DUBs with minimal effects on the function of 20S proteasome; (iv) Au(PPh3)PT induced apoptosis in cancer cells from acute myeloid leukemia patients; (v) Au(PPh3)PT effectively suppressed the growth of lung adenocarcinoma xenografts in nude mice; and (vi) Au (PPh3)PT elicited less cytotoxicity in normal cells than several other metal compounds. INTERPRETATION: Together, this study discovers a new gold(I) complex to be an effective inhibitor of the DUBs and a potential anti-cancer drug. FUND: The National High Technology Research and Development Program of China, the project of Guangdong Province Natural Science Foundation, the projects from Foundation for Higher Education of Guangdong, the project from Guangzhou Medical University for Doctor Scientists, the Medical Scientific Research Foundation of Guangdong Province, and the Guangzhou Key Medical Discipline Construction Project Fund.

A novel deubiquitinase inhibitor b-AP15 triggers apoptosis in both androgen receptor-dependent and -independent prostate cancers.

Prostate cancer (PCa) remains a leading cause of cancer-related death in men. Especially, a subset of patients will eventually progress to the metastatic castrate-resistant prostate cancer (CRPC) which is currently incurable. Deubiquitinases (DUBs) associated with the 19S proteasome regulatory particle are increasingly emerging as significant therapeutic targets in numerous cancers. Recently, a novel small molecule b-AP15 is identified as an inhibitor of the USP14/UCHL5 (DUBs) of the 19S proteasome, resulting in cell growth inhibition and apoptosis in several human cancer cell lines. Here, we studied the therapeutic effect of b-AP15 in PCa, and our results indicate that (i) b-AP15 decreases viability, proliferation and triggers cytotoxicity to both androgen receptor-dependent and -independent PCa cells in vitro and in vivo, associated with caspase activation, inhibition of mitochondria function, increased reactive oxygen species (ROS) generation and endoplasmic reticulum (ER) stress; (ii) pan-caspase inhibitor z-VAD-FMK and ROS scavenger N-acetyl-L-cysteine (NAC) efficiently block apoptosis but not proteasome inhibition induced by exposure of b-AP15; (iii) treatment with b-AP15 in androgen-dependent prostate cancer (ADPC) cells down-regulates the expression of androgen receptor (AR), which is degraded via the ubiquitin proteasome system. Hence, the potent anti-tumor effect of b-AP15 on both androgen receptor-dependent and -independent PCa cells identifies a new promising therapeutic strategy for prostate cancer.

Platinum pyrithione induces apoptosis in chronic myeloid leukemia cells resistant to imatinib via DUB inhibition-dependent caspase activation and Bcr-Abl downregulation.

Chronic myelogenous leukemia (CML) is characterized by the chimeric tyrosine kinase Bcr-Abl. T315I Bcr-Abl is the most notorious point mutation to elicit acquired resistance to imatinib (IM), leading to poor prognosis. Therefore, it is urgent to search for additional approaches and targeting strategies to overcome IM resistance. We recently reported that platinum pyrithione (PtPT) potently inhibits the ubiquitin-proteasome system (UPS) via targeting the 26 S proteasome-associated deubiquitinases (DUBs), without effecting on the 20 S proteasome. Here we further report that (i) PtPT induces apoptosis in Bcr-Abl wild-type and Bcr-Abl-T315I mutation cells including the primary mononuclear cells from CML patients clinically resistant to IM, as well as inhibits the growth of IM-resistant Bcr-Abl-T315I xenografts in vivo; (ii) PtPT downregulates Bcr-Abl level through restraining Bcr-Abl transcription, and decreasing Bcr-Abl protein mediated by DUBs inhibition-induced caspase activation; (iii) UPS inhibition is required for PtPT-induced caspase activation and cell apoptosis. These findings support that PtPT overcomes IM resistance through both Bcr-Abl-dependent and -independent mechanisms. We conclude that PtPT can be a lead compound for further drug development to overcome imatinib resistance in CML patients.

Nickel pyrithione induces apoptosis in chronic myeloid leukemia cells resistant to imatinib via both Bcr/Abl-dependent and Bcr/Abl-independent mechanisms.

BACKGROUND: Acquired imatinib (IM) resistance is frequently characterized by Bcr-Abl mutations that affect IM binding and kinase inhibition in patients with chronic myelogenous leukemia (CML). Bcr-Abl-T315I mutation is the predominant mechanism of the acquired resistance to IM. Therefore, it is urgent to search for additional approaches and targeting strategies to overcome IM resistance. We recently reported that nickel pyrithione (NiPT) potently inhibits the ubiquitin proteasome system via targeting the 19S proteasome-associated deubiquitinases (UCHL5 and USP14), without effecting on the 20S proteasome. In this present study, we investigated the effect of NiPT, a novel proteasomal deubiquitinase inhibitor, on cell survival or apoptosis in CML cells bearing Bcr-Abl-T315I or wild-type Bcr-Abl. METHODS: Cell viability was examined by MTS assay and trypan blue exclusion staining assay in KBM5, KBM5R, K562, BaF3-p210-WT, BaF3-p210-T315I cells, and CML patients' bone marrow samples treated with NiPT. Cell apoptosis in CML cells was detected with Annexin V-FITC/PI and rhodamine-123 staining followed by fluorescence microscopy and flow cytometry and with western blot analyses for apoptosis-associated proteins. Expression levels of Bcr-Abl in CML cells were analyzed by using western blotting and real-time PCR. The 20S proteasome peptidase activity was measured using specific fluorogenic substrate. Active-site-directed labeling of proteasomal DUBs, as well as the phosphorylation of USP14 was used for evaluating the inhibition of the DUBs activity by NiPT. Mouse xenograft models of KBM5 and KBM5R cells were analyzed, and Bcr-Abl-related proteins and protein biomarkers related to proliferation, differentiation, and adhesion in tumor tissues were detected by western blots and/or immunohistological analyses. RESULTS: NiPT induced apoptosis in CML cells and inhibited the growth of IM-resistant Bcr-Abl-T315I xenografts in nude mice. Mechanistically, NiPT induced decreases in Bcr-Abl proteins, which were associated with downregulation of Bcr-Abl transcription and with the cleavage of Bcr-Abl protein by activated caspases. NiPT-induced ubiquitin proteasome system inhibition induced caspase activation in both IM-resistant and IM-sensitive CML cells, and the caspase activation was required for NiPT-induced Bcr-Abl downregulation and apoptotic cell death. CONCLUSIONS: These findings support that NiPT can overcome IM resistance through both Bcr-Abl-dependent and Bcr-Abl-independent mechanisms, providing potentially a new option for CML treatment.

Two clinical drugs deubiquitinase inhibitor auranofin and aldehyde dehydrogenase inhibitor disulfiram trigger synergistic anti-tumor effects in vitro and in vivo.

Inhibition of proteasome-associated deubiquitinases (DUBs) is emerging as a novel strategy for cancer therapy. It was recently reported that auranofin (Aur), a gold (I)-containing compound used clinically to treat rheumatoid arthritis, is a proteasome-associated DUB inhibitor. Disulfiram (DSF), an inhibitor of aldehyde dehydrogenase, is currently in clinical use for treating alcoholism. Recent studies have indicated that DSF can also act as an antitumor agent. We investigated the effect of combining DSF and Aur on apoptosis induction and tumor growth in hepatoma cancer cells. Here we report that (i) the combined treatment of Aur and DSF results in synergistic cytotoxicity to hepatoma cells in vitro and in vivo; (ii) Aur and DSF in combination induces caspase activation, endoplasmic reticulum (ER) stress, and reactive oxygen species (ROS) production; (iii) pan-caspase inhibitor z-VAD-FMK could efficiently block apoptosis but not proteasome inhibition induced by Aur and DSF combined treatment, and ROS is not required for Aur+DSF to induce apoptosis. Collectively, we demonstrate a model of synergism between DSF and proteasome-associated DUB inhibitor Aur in the induction of apoptosis in hepatoma cancer cells, identifying a potential novel anticancer strategy for clinical use in the future.