Evaluation of a two-test strategy for HIV screening in a low-prevalence setting and the indications for optimizing clinical management.

Objectives: To evaluate a two-test strategy for HIV screening in the low-prevalence population and to assess the feasibility of utilizing the optimal signal-to-cutoff (S/CO) threshold on the chemiluminescence immunoassay(CMIA) and an additional rapid test on the gold immune-chromatography assay (GICA) for screening positive patients and optimization of clinical management. Methods: We conducted a retrospective study of samples analyzed by the fourth-generation Architect HIV Ag/Ab combo assay (CMIA) in a large medical center between June 2017 and August 2020. Reactive samples underwent a second screening test using the rapid test GICA, followed by Western blot (WB) as the confirmatory test. Receiver operating characteristic (ROC) curve analysis was used to determine the optimal S/CO. We calculated sensitivity, specificity, and predictive value based on our population. The performance of the single-test strategy (CMIA) was compared with that of the two-test strategy (CMIA and GICA). Logistic regression was used to analyze the factors of clinical characteristics leading to false positive results. Results: A total of 220558 samples were screened by CMIA, and 429 patients met the inclusion criteria. Of these, CMIA produced 199 false-positive results with a median S/CO of 1.93(IQR1.45-3.68) and 230 positive results with a median S/CO of 455.1 (IQR169.3-709.7). The optimal S/CO of the single-test strategy was 8.82, which achieved a sensitivity of 100% and a positive predictive value (PPV) of 90.9%. The two-test strategy (CMIA and GICA) provided a sensitivity of 100% and a PPV of 98.7%, which best correlated with the confirmatory test WB. The combination of S/CO 8.82 on the CMIA assay and additional test results of GICA can be defined as four types used to interpret HIV serostatus. The false positive rate (FPR) was high in the female, the age≤18 group, the pre-operative patients, and the patients from the clinical departments of Pediatrics, Gynecology and Obstetrics, and Oncology, etc. Conclusions: The false positive rate is high in the low-prevalence setting by using CMIA. The two-test strategy (CMIA and GICA) is recommended for HIV screening in hospitals. Hopefully, the clinicians will be able to interpret HIV serostatus and facilitate clinical decision-making while waiting for the confirmatory results.

Piperlongumine overcomes imatinib resistance by inducing proteasome inhibition in chronic myelogenous leukemia cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Piper longum L., an herbal medicine used in India and other Asian countries, is prescribed routinely for a range of diseases, including tumor. Piperlongumine, a natural product isolated from Piper longum L., has received widespread attention due to its various pharmacological activities, such as anti-inflammatory, antimicrobial, and antitumor effects. AIM OF THE STUDY: Chronic myelogenous leukemia (CML) is a hematopoietic disease caused by Bcr-Abl fusion gene, with an incidence of 15% in adult leukemias. Targeting Bcr-Abl by imatinib provides a successful treatment approach for CML. However, imatinib resistance is an inevitable issue for CML treatment. In particular, T315I mutant is the most stubborn of the Bcr-Abl point mutants associated with imatinib resistance. Therefore, it is urgent to find an alternative approach to conquer imatinib resistance. This study investigated the role of a natural product piperlongumine in overcoming imatinib resistance in CML. MATERIALS AND METHODS: Cell viability and apoptosis were evaluated by MTS assay and Annexin V/propidium iodide counterstaining assay, respectively. Levels of intracellular signaling proteins were assessed by Western blots. Mitochondrial membrane potential was reflected by the fluorescence intensity of rhodamine-123. The function of proteasome was detected using 20S proteasomal activity assay, proteasomal deubiquitinase activity assay, and deubiquitinase active-site-directed labeling. The antitumor effects of piperlongumine were assessed with mice xenografts. RESULTS: We demonstrate that (i) Piperlongumine inhibits proteasome function by targeting 20S proteasomal peptidases and 19S proteasomal deubiquitinases (USP14 and UCHL5) in Bcr-Abl-WT and Bcr-Abl-T315I CML cells; (ii) Piperlongumine inhibits the cell viability of CML cell lines and primary CML cells; (iii) Proteasome inhibition by piperlongumine leads to cell apoptosis and downregulation of Bcr-Abl; (iv) Piperlongumine suppresses the tumor growth of CML xenografts. CONCLUSIONS: These results support that blockade of proteasome activity by piperlongumine provides a new therapeutic strategy for treating imatinib-resistant CML.

Physiological and Population Responses of Nilaparvata lugens after Feeding on Drought-Stressed Rice.

Drought stress greatly impacts insect development and population growth. Some studies have demonstrated increased reproductive capacity in drought-stressed insects; however, physiological changes in the brown planthopper (BPH), Nilaparvata lugens (Stål), during periods of drought are unclear. In this study, BPH fed on drought- stressed rice had lower population numbers than BPH feeding on non-stressed rice. Water content, osmotic pressure of hemolymph and total amino acid content of BPH were significantly lower when BPH fed on drought-stressed rice compared to the non-stressed control; however, glucose content and glutathione S-transferase (GST) activity were significantly higher in BPH fed on drought-stressed rice. The expression of Vitellogenin and Exuperantia in BPH fed on drought-stressed rice was higher than that in BPH feeding on non-stressed control plants. The size of myofibrils and the abundance of mitochondria in BPH flight muscles were significantly lower in BPH fed on drought-stressed rice compared to non-stressed plants. These results indicate that water management impacts the physiology of BPH, which may be useful in understanding the relationship between drought stress and this damaging herbivore.

Pseudolaric acid B induces mitotic arrest and apoptosis in both imatinib-sensitive and -resistant chronic myeloid leukaemia cells.

The selective BCR-ABL tyrosine kinase inhibitor imatinib is one of the first-line therapies in the management of chronic myeloid leukaemia (CML). However, acquired resistance to this inhibitor, which is especially conferred by the T315I point mutation in BCR-ABL, impedes the efficacy of imatinib therapy. Therefore, the discovery and development of novel agents to overcome imatinib resistance is urgently needed. Pseudolaric acid B (PAB), a small molecule isolated from the traditional Chinese medicine Cortex pseudolaricis, has been reported to be a potential candidate for immune disorders and cancer treatment. However, its effects on CML and the involved molecular mechanism have not been reported. In the current study, by performing both in vitro and in vivo experiments in CML cells, we showed that PAB blocked the cell cycle at G2/M phase and subsequently activated the caspase pathway, cleaved the BCR-ABL protein and inhibited the BCR-ABL downstream pathways, ultimately leading to cell proliferation inhibition, cytotoxicity and apoptosis. These events were observed in both imatinib-sensitive and imatinib-insensitive CML cell lines. Moreover, PAB decreased the viability of primary blood mononuclear cells from CML patients and induced apoptosis in these cells. Our findings suggest that PAB could be used as a novel agent to sensitize imatinib-resistant CML.

Proteasomal cysteine deubiquitinase inhibitor b-AP15 suppresses migration and induces apoptosis in diffuse large B cell lymphoma.

BACKGROUND: The first line therapy for patients with diffuse large B cell (DLBCL) is R-CHOP. About half of DLBCL patients are either refractory to, or will relapse, after the treatment. Therefore, identifying novel drug targets and effective therapeutic agents is urgently needed for improving DLBCL patient survival. b-AP15, a selective small molecule inhibitor of proteasomal USP14 and UCHL5 deubiquitinases (DUBs), has shown selectivity and efficacy in several other types of cancer cells. This is the first study to report the effect of b-AP15 in DLBCL. METHODS: Cell lines of two DLBCL subtypes, Germinal Center B Cell/ GCB (SU-DHL-4, OCI-LY-1, OCI-LY-19) and Activated B Cell/ABC (SU-DHL-2), were used in the current study. Cell viability was measured by MTS assay, proliferation by trypan blue exclusion staining assay, cellular apoptosis by Annexin V-FITC/PI staining and mitochondrial outer membrane permeability assays, the activities of 20S proteasome peptidases by cleavage of specific fluorogenic substrates, and cell migration was detected by transwell assay in these GCB- and ABC-DLBCL cell lines. Mouse xenograft models of SU-DHL-4 and SU-DHL-2 cells were used to determine in vivo effects of b-AP15 in DLBCL tumors. RESULTS: b-AP15 inhibited proteasome DUB activities and activated cell death pathway, as evident by caspase activation and mitochondria apoptosis in GCB- and ABC- DLBCL cell lines. b-AP15 treatment suppressed migration of GCB- and ABC-DLBCL cells via inhibiting Wnt/ß-catenin and TGFß/Smad pathways. Additionally, b-AP15 significantly inhibited the growth of GCB- and ABC DLBCL in xenograft models. CONCLUSIONS: These results indicate that b-AP15 inhibits cell migration and induces apoptosis in GCB- and ABC-DLBCL cells, and suggest that inhibition of 19S proteasomal DUB should be a novel strategy for DLBCL treatment.

Cytoplasmic RAP1 mediates cisplatin resistance of non-small cell lung cancer.

Cytotoxic chemotherapy agents (e.g., cisplatin) are the first-line drugs to treat non-small cell lung cancer (NSCLC) but NSCLC develops resistance to the agent, limiting therapeutic efficacy. Despite many approaches to identifying the underlying mechanism for cisplatin resistance, there remains a lack of effective targets in the population that resist cisplatin treatment. In this study, we sought to investigate the role of cytoplasmic RAP1, a previously identified positive regulator of NF-κB signaling, in the development of cisplatin resistance in NSCLC cells. We found that the expression of cytoplasmic RAP1 was significantly higher in high-grade NSCLC tissues than in low-grade NSCLC; compared with a normal pulmonary epithelial cell line, the A549 NSCLC cells exhibited more cytoplasmic RAP1 expression as well as increased NF-κB activity; cisplatin treatment resulted in a further increase of cytoplasmic RAP1 in A549 cells; overexpression of RAP1 desensitized the A549 cells to cisplatin, and conversely, RAP1 depletion in the NSCLC cells reduced their proliferation and increased their sensitivity to cisplatin, indicating that RAP1 is required for cell growth and has a key mediating role in the development of cisplatin resistance in NSCLC cells. The RAP1-mediated cisplatin resistance was associated with the activation of NF-κB signaling and the upregulation of the antiapoptosis factor BCL-2. Intriguingly, in the small portion of RAP1-depleted cells that survived cisplatin treatment, no induction of NF-κB activity and BCL-2 expression was observed. Furthermore, in established cisplatin-resistant A549 cells, RAP1 depletion caused BCL2 depletion, caspase activation and dramatic lethality to the cells. Hence, our results demonstrate that the cytoplasmic RAP1-NF-κB-BCL2 axis represents a key pathway to cisplatin resistance in NSCLC cells, identifying RAP1 as a marker and a potential therapeutic target for cisplatin resistance of NSCLC.

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.

Repurposing an antidandruff agent to treating cancer: zinc pyrithione inhibits tumor growth via targeting proteasome-associated deubiquitinases.

The ubiquitin-proteasome system (UPS) plays a central role in various cellular processes through selectively degrading proteins involved in critical cellular functions. Targeting UPS has been validated as a novel strategy for treating human cancer, as inhibitors of the 20S proteasome catalytic activity are currently in clinical use for treatment of multiple myeloma and other cancers, and the deubiquitinase activity associated with the proteasome is also a valid target for anticancer agents. Recent studies suggested that zinc pyrithione, an FDA-approved antidandruff agent, may have antitumor activity, but the detailed molecular mechanisms remain unclear. Here we report that zinc pyrithione (ZnPT) targets the proteasome-associated DUBs (USP14 and UCHL5) and inhibits their activities, resulting in a rapid accumulation of protein-ubiquitin conjugates, but without inhibiting the proteolytic activities of 20S proteasomes. Furthermore, ZnPT exhibits cytotoxic effects against various cancer cell lines in vitro, selectively kills bone marrow cells from leukemia patients ex vivo, and efficiently inhibits the growth of lung adenocarcinoma cancer cell xenografts in nude mice. This study has identified zinc pyrithione, an FDA-approved pharmacological agent with potential antitumor properties as a proteasomal DUB inhibitor.

Bilirubin neurotoxicity is associated with proteasome inhibition.

The molecular mechanism underlying bilirubin neurotoxicity remains obscure. Ubiquitin-proteasome system-mediated proteolysis is pivotal to virtually all cellular processes and cell survival. Here we report for the first time that bilirubin at a clinically relevant elevated level impairs proteasomal function via inhibiting both the 19S proteasome-associated deubiquitinases (USP14 and UCHL5) and the chymotrypsin-like (CT-like) peptidase activity of 20S proteasomes, thereby contributing to bilirubin neurotoxicity. This is supported by multiple lines of evidence. First, sera from patients with hyperbilirubinemia were able to inhibit the peptidase activity of purified 20S proteasome in vitro in a bilirubin concentration-dependent manner; meanwhile, the blood cells of these patients showed significantly increased levels of ubiquitinated proteins (Ub-prs), consistent with proteasome inhibition. Second, intracerebroventricular injection to adult rats or intraperitoneal injections to neonatal rats of bilirubin-induced neural accumulation of Ub-prs, concurrent with other neural pathology; and brain malfunction and pathology induced by neonatal exposure to hyperbilirubinemia were detectable in the rats during their adulthood. Third, in primary cultures of hippocampal neurons, bilirubin strikingly induced Ub-pr accumulation before the activation of cell death pathway becomes discernible. Finally, bilirubin in vitro directly inhibited both the deubiquitination activity of proteasome-associated USP14 and UCHL5 and the CT-like peptidase activity of purified 20S proteasomes, in a dose-dependent manner. Hence, this study has discovered that increased bilirubin at a clinically achievable level can act as a proteasome inhibitor via targeting the 19S proteasome-associated deubiquitinases (DUBs) and, perhaps to a less extent, the 20S proteasome, identifying a novel mechanism for bilirubin neurotoxicity.

Platinum-containing compound platinum pyrithione is stronger and safer than cisplatin in cancer therapy.

DNA is the well-known molecular target of current platinum-based anticancer drugs; consequently, their clinical use is severely restricted by their systemic toxicities and drug resistance originating from non-selective DNA damage. Various strategies have been developed to circumvent the shortcomings of platinum-based chemotherapy but the inherent problem remains unsolved. Here we report that platinum pyrithione (PtPT), a chemically well-characterized synthetic complex of platinum, inhibits proteasome function and thereby exhibits greater and more selective cytotoxicity to multiple cancer cells than cisplatin, without showing discernible DNA damage both in vitro and in vivo. Moreover, unlike the classical proteasome inhibitor bortezomib/Velcade which inhibits the proteasome via blocking the peptidase activity of 20S proteasomes, PtPT primarily deactivates 26S proteasome-associated deubiquitinases USP14 and UCHL5. Furthermore, PtPT can selectively induce cytotoxicity and proteasome inhibition in cancer cells from leukemia patients but not peripheral blood mononuclear cells from healthy humans. In nude mice, PtPT also remarkably inhibited tumor xenograft growth, without showing the adverse effects that were induced by cisplatin. Hence, we have discovered a new platinum-based anti-tumor agent PtPT which targets 26S proteasome-associated deubiquitinases rather than DNA in the cell and thereby exerts safer and more potent anti-tumor effects, identifying a highly translatable new platinum-based anti-cancer strategy.

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.

A microRNA-mediated decrease in eukaryotic initiation factor 2α promotes cell survival during PS-341 treatment.

MicroRNAs (miRs) play pivotal roles in carcinogenesis and endoplasmic reticulum (ER) that performs the folding, modification and trafficking of proteins targeted to the secretory pathway. Cancer cells often endure ER stress during tumor progression but use the adaptive ER stress response to gain survival advantage. Here we report: (i) A group of miRs, including miR-30b-5p and miR-30c-5p, are upregulated by proteasome inhibitor PS-341 treatment, in HepG2 and MDA-MB-453 cells. (ii) Two representative PS-341-induced miRs: miR-30b-5p and miR-30c-5p are found to promote cell proliferation and anti-apoptosis in both tumor cells. (iii) eIF2α is confirmed as the congenerous target of miR-30b-5p and miR-30c-5p, essential to the anti-apoptotic function of these miRs. (iv) Upregulation of miR-30b-5p or miR-30c-5p, which occurs latter than the increase of phosphorylated eIF2α (p-eIF2α) in the cell under ER stress, suppresses the p-eIF2α/ATF4/CHOP pro-apoptotic pathway. (v) Inhibition of the miR-30b-5p or miR-30c-5p sensitizes the cancer cells to the cytotoxicity of proteasome inhibition. In conclusion, we unravels a new miRs-based mechanism that helps maintain intracellular proteostasis and promote cell survival during ER stress through upregulation of miR-30b-5p and miR-30c-5p which target eIF2α and thereby inhibit the p-eIF2α/ATF4/CHOP pro-apoptotic pathway, identifying miR-30b-5p and miR-30c-5p as potentially new targets for anti-cancer therapies.

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.

Gambogic acid induces apoptosis in diffuse large B-cell lymphoma cells via inducing proteasome inhibition.

Resistance to chemotherapy is a great challenge to improving the survival of patients with diffuse large B-cell lymphoma (DLBCL), especially those with activated B-cell-like DLBCL (ABC-DLBCL). Therefore it is urgent to search for novel agents for the treatment of DLBCL. Gambogic acid (GA), a small molecule derived from Chinese herb gamboges, has been approved for Phase II clinical trial for cancer therapy by Chinese FDA. In the present study, we investigated the effect of GA on cell survival and apoptosis in DLBCL cells including both GCB- and ABC-DLBCL cells. We found that GA induced growth inhibition and apoptosis of both GCB- and ABC-DLBCL cells in vitro and in vivo, which is associated with proteasome malfunction. These findings provide significant pre-clinical evidence for potential usage of GA in DLBCL therapy particularly in ABC-DLBCL treatment.

Anacardic acid induces cell apoptosis associated with induction of ATF4-dependent endoplasmic reticulum stress.

Anacardic acid (6-pentadecylsalicylic acid, AA), a natural compound isolated from the traditional medicine Amphipterygium adstringens, has been reported to possess antitumor activities. However, its molecular targets have not been thoroughly studied. Here, we report that AA is a potent inducer of endoplasmic reticulum (ER) stress, leading to apoptosis in hepatoma HepG2 and myeloma U266 cells. Induction of ER stress by AA was supported by a dose- and time-dependent increase in expression of the ER signaling downstream molecules, such as GRP78/BiP, phosphorylated eIF2α, ATF4 and CHOP in both HepG2 and U266 cell lines. Blockage of ATF4 expression by siRNA partially inhibited, while knockdown of CHOP expression by siRNA slightly increased AA-induced cell death in these cells. In addition, AA suppressed HepG2 xenograft tumor growth, associated with increased ER stress in vivo. These results suggest that AA induces tumor cell apoptosis associated with ATF4-dependent ER stress.

Gambogic acid induces apoptosis in imatinib-resistant chronic myeloid leukemia cells via inducing proteasome inhibition and caspase-dependent Bcr-Abl downregulation.

PURPOSE: Chronic myelogenous leukemia (CML) is characterized by the constitutive activation of Bcr-Abl tyrosine kinase. Bcr-Abl-T315I is the predominant mutation that causes resistance to imatinib, cytotoxic drugs, and the second-generation tyrosine kinase inhibitors. The emergence of imatinib resistance in patients with CML leads to searching for novel approaches to the treatment of CML. Gambogic acid, a small molecule derived from Chinese herb gamboges, has been approved for phase II clinical trial for cancer therapy by the Chinese Food and Drug Administration (FDA). In this study, we investigated the effect of gambogic acid on cell survival or apoptosis in CML cells bearing Bcr-Abl-T315I or wild-type Bcr-Abl. EXPERIMENTAL DESIGN: CML cell lines (KBM5, KBM5-T315I, and K562), primary cells from patients with CML with clinical resistance to imatinib, and normal monocytes from healthy volunteers were treated with gambogic acid, imatinib, or their combination, followed by measuring the effects on cell growth, apoptosis, and signal pathways. The in vivo antitumor activity of gambogic acid and its combination with imatinib was also assessed with nude xenografts. RESULTS: Gambogic acid induced apoptosis and cell proliferation inhibition in CML cells and inhibited the growth of imatinib-resistant Bcr-Abl-T315I xenografts in nude mice. Our data suggest that GA-induced proteasome inhibition is required for caspase activation in both imatinib-resistant and -sensitive CML cells, and caspase activation is required for gambogic acid-induced Bcr-Abl downregulation and apoptotic cell death. CONCLUSIONS: These findings suggest an alternative strategy to overcome imatinib resistance by enhancing Bcr-Abl downregulation with the medicinal compound gambogic acid, which may have great clinical significance in imatinib-resistant cancer therapy.

Anti-rheumatic agent auranofin induced apoptosis in chronic myeloid leukemia cells resistant to imatinib through both Bcr/Abl-dependent and -independent mechanisms.

Resistance to Imatinib mesylate (IM) is an emerging problem for patients with chronic myelogenous leukemia (CML). T315I mutation in the Bcr-Abl is the predominant mechanism of the acquired resistance to IM and second generation tyrosine kinase inhibitors (TKI). Therefore it is urgent to search for new measures to overcome TKI-resistance. Auranofin (AF), clinically used to treat rheumatic arthritis, was recently approved by US Food and Drug Administration for Phase II clinical trial to treat cancer. In contrast to the reports that AF induces apoptosis by increasing intracellular reactive oxygen species (ROS) levels via inhibiting thioredoxin reductase, our recent study revealed that AF-induced apoptosis depends on inhibition of proteasomal deubiquitinases (UCHL5 and USP14). Here we report that (i) AF induces apoptosis in both Bcr-Abl wild-type cells and Bcr-Abl-T315I mutation cells and inhibits the growth of IM-resistant Bcr-Abl-T315I xenografts in vivo; (ii) AF inhibits Bcr-Abl through both downregulation of Bcr-Abl gene expression and Bcr-Abl cleavage mediated by proteasome inhibition-induced caspase activation; (iii) proteasome inhibition but not ROS is required for AF-induced caspase activation and apoptosis. These findings support that AF overcomes IM resistance through both Bcr/Abl-dependent and -independent mechanisms, providing great clinical significance for cancer treatment.

A novel proteasome inhibitor suppresses tumor growth via targeting both 19S proteasome deubiquitinases and 20S proteolytic peptidases.

The successful development of bortezomib-based therapy for treatment of multiple myeloma has established proteasome inhibition as an effective therapeutic strategy, and both 20S proteasome peptidases and 19S deubiquitinases (DUBs) are becoming attractive targets of cancer therapy. It has been reported that metal complexes, such as copper complexes, inhibit tumor proteasome. However, the involved mechanism of action has not been fully characterized. Here we report that (i) copper pyrithione (CuPT), an alternative to tributyltin for antifouling paint biocides, inhibits the ubiquitin-proteasome system (UPS) via targeting both 19S proteasome-specific DUBs and 20S proteolytic peptidases with a mechanism distinct from that of the FDA-approved proteasome inhibitor bortezomib; (ii) CuPT potently inhibits proteasome-specific UCHL5 and USP14 activities; (iii) CuPT inhibits tumor growth in vivo and induces cytotoxicity in vitro and ex vivo. This study uncovers a novel class of dual inhibitors of DUBs and proteasome and suggests a potential clinical strategy for cancer therapy.

Clinically used antirheumatic agent auranofin is a proteasomal deubiquitinase inhibitor and inhibits tumor growth.

Proteasomes are attractive emerging targets for anti-cancer therapies. Auranofin (Aur), a gold-containing compound clinically used to treat rheumatic arthritis, was recently approved by US Food and Drug Administration for Phase II clinical trial to treat cancer but its anti-cancer mechanism is poorly understood. Here we report that (i) Aur shows proteasome-inhibitory effect that is comparable to that of bortezomib/Velcade (Vel); (ii) different from bortezomib, Aur inhibits proteasome-associated deubiquitinases (DUBs) UCHL5 and USP14 rather than the 20S proteasome; (iii) inhibition of the proteasome-associated DUBs is required for Aur-induced cytotoxicity; and (iv) Aur selectively inhibits tumor growth in vivo and induces cytotoxicity in cancer cells from acute myeloid leukemia patients. This study provides important novel insight into understanding the proteasome-inhibiting property of metal-containing compounds. Although several DUB inhibitors were reported, this study uncovers the first drug already used in clinic that can inhibit proteasome-associated DUBs with promising anti-tumor effects.