[Show abstract][Hide abstract] ABSTRACT: The ubiquitin-proteasome system has recently been implicated in various pathologies including neurodegenerative diseases and cancer. In light of this, techniques for studying the regulatory mechanism of this system are essential to elucidating the cellular and molecular processes of the aforementioned diseases. The use of hemagglutinin derived ubiquitin probes outlined in this paper serves as a valuable tool for the study of this system. This paper details a method that enables the user to perform assays that give a direct visualization of deubiquitinating enzyme activity. Deubiquitinating enzymes control proteasomal degradation and share functional homology at their active sites, which allows the user to investigate the activity of multiple enzymes in one assay. Lysates are obtained through gentle mechanical cell disruption and incubated with active site directed probes. Functional enzymes are tagged with the probes while inactive enzymes remain unbound. By running this assay, the user obtains information on both the activity and potential expression of multiple deubiquitinating enzymes in a fast and easy manner. The current method is significantly more efficient than using individual antibodies for the predicted one hundred deubiquitinating enzymes in the human cell.
Journal of Visualized Experiments 05/2015; DOI:10.3791/52784 · 1.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Breast cancer is one of the leading causes of cancer death among women in the United States. Patients expressing the estrogen and progesterone receptor (ER and PR) and human epidermal growth factor 2 (HER-2) tumor markers have favorable prognosis and efficacious therapeutic options. In contrast, tumors that are negative for these markers (triple-negative) have a disproportionate share of morbidity and mortality due to lack of a validated molecular target.Deubiquitinating enzymes (DUBs) are a critical component of ubiquitin-proteasome-system degradation and have been shown to be differentially expressed and activated in a number of cancers, including breast, with their aberrant activity linked to cancer prognosis and clinical outcome. We evaluated the effect of the DUB inhibitors b-AP15 and RA-9 alone and in combination with early- and late-stage lysosomal inhibitors on cell viability in a panel of triple negative breast cancer (TNBC) cell lines.Our results indicate small-molecule DUB inhibitors have a profound effect on TNBC viability and lead to activation of autophagy as a cellular mechanism to compensate for ubiquitin-proteasome-system stress. Treatment with sub-optimal doses of DUB and lysosome inhibitors synergistically kills TNBC cells. This supports the evaluation of DUB inhibition, in combination with lysosomal inhibition, as a therapeutic approach for the treatment of TNBC.
[Show abstract][Hide abstract] ABSTRACT: Ovarian cancer is the deadliest of the gynecological malignancies. Carcinogenic progression is accompanied by up-regulation of ubiquitin-dependent protein degradation machinery as a mechanism to compensate with elevated endogenous proteotoxic stress. Recent studies support the notion that deubiquitinating enzymes (DUBs) are essential factors in proteolytic degradation and that their aberrant activity is linked to cancer progression and chemoresistance. Thus, DUBs are an attractive therapeutic target for ovarian cancer.
The potency and selectivity of RA-9 inhibitor for proteasome-associated DUBs was determined in ovarian cancer cell lines and primary cells. The anticancer activity of RA-9 and its mechanism of action was evaluated in multiple cancer cell lines in vitro and in vivo in immunodeficient mice bearing an intra-peritoneal ES-2 xenograft model of human ovarian cancer.
Here we report the characterization of RA-9 as a small-molecule inhibitor of proteasome-associated DUBs. Treatment with RA-9 selectively induces onset of apoptosis, in ovarian cancer cell lines and primary cultures derived from donors. Loss of cell viability following RA-9 exposure is associated with an Unfolded Protein Response (UPR) as mechanism to compensate for unsustainable levels of proteotoxic stress. In vivo treatment with RA-9 retards tumor growth, increases overall survival and was well tolerated by the host.
Our preclinical studies support further evaluation of RA-9 as an ovarian cancer therapeutic.
Clinical Cancer Research 04/2014; 20(12). DOI:10.1158/1078-0432.CCR-13-2658 · 8.72 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a valuable tool for understanding ovarian cancer, their use has many limitations. These include the lack of heterogeneity and the plethora of genetic alterations associated with extended in vitro passaging. Here we describe a method that allows for rapid establishment of primary ovarian cancer cells form solid clinical specimens collected at the time of surgery. The method consists of subjecting clinical specimens to enzymatic digestion for 30 min. The isolated cell suspension is allowed to grow and can be used for downstream application including drug screening. The advantage of primary ovarian cancer cell lines over established ovarian cancer cell lines is that they are representative of the original specific clinical specimens they are derived from and can be derived from different sites whether primary or metastatic ovarian cancer.
Journal of Visualized Experiments 02/2014; DOI:10.3791/51581 · 1.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ovarian cancer is the deadliest of the gynecological diseases and the fifth cause of cancer death among American women. This is mainly due to the lack of prognostic tools capable of detecting early stages of ovarian cancer and to the high rate of resistance to the current chemotherapeutic regimens. In this scenario the overall 5-year survival rate for ovarian cancer patients diagnosed at late stage is less than 25%. Abnormalities associated with the malignant phenotype and the mechanisms of tumor progression are not clearly understood. In vitro studies are necessary, yet have been hampered due to the limitations accompanied with the use of ovarian cancer cell lines and the heterogeneity of the ovarian cancer cell population derived from ascites fluids. In this study we present a simple, rapid and reproducible method for the isolation and characterization of ovarian cancer cells from solid tumor tissue and show that enzymatic digestion for 30 minutes with dispase II results in the most effective recovery of viable epithelial ovarian cancer (EOC) cells. The resulting cancer (EOC) cell preparations demonstrate a significant yield, high levels of viability and are fibroblast-free. They grow for up to six passages and retain the capacity of forming spheroids-like structures in agarose. In addition, they can be genetically manipulated and used for drug screening, thus rendering them highly suitable for downstream applications. Notably, isolation of ovarian cancer cells from solid specimens using this method has the advantage of allowing for isolation of cancer cells from early stages of ovarian cancer as well as obtaining cells from defined either primary and/or metastatic ovarian cancer sites. Thus, these cells are highly suitable for investigations aimed at understanding ovarian cancer.
PLoS ONE 11/2012; 7(11):e50519. DOI:10.1371/journal.pone.0050519 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cervical cancer cells exhibit an increased requirement for ubiquitin-dependent protein degradation associated with an elevated metabolic turnover rate, and for specific signaling pathways, notably HPV E6-targeted degradation of p53 and PDZ proteins. Natural compounds with antioxidant properties including flavonoids and triterpenoids hold promise as anticancer agents by interfering with ubiquitin-dependent protein degradation. An increasing body of evidence indicates that their α-β unsaturated carbonyl system is the molecular determinant for inhibition of ubiquitin-mediated protein degradation up-stream of the catalytic sites of the 20S proteasome. Herein we report the identification and characterization of a new class of chalcone-based, potent and cell permeable chemical inhibitors of ubiquitin-dependent protein degradation, and a lead compound RAMB1. RAMB1 inhibits ubiquitin-dependent protein degradation without compromising the catalytic activities of the 20S proteasome, a mechanism distinct from that of Bortezomib. Treatment of cervical cancer cells with RAMB1 triggers unfolded protein responses, including aggresome formation and Hsp90 stabilization, and increases p53 steady state levels. RAMB1 treatment results in activation of lysosomal-dependent degradation pathways as a mechanism to compensate for increasing levels of poly-ubiquitin enriched toxic aggregates. Importantly, RAMB1 synergistically triggers cell death of cervical cancer cells when combined with the lysosome inhibitor Chloroquine.
PLoS ONE 08/2011; 6(8):e23888. DOI:10.1371/journal.pone.0023888 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Proteasome inhibitors have potential for the treatment of cervical cancer. We describe the synthesis and biological characterization of a new series of 1,3-diphenylpropen-1-one (chalcone) based derivatives lacking the boronic acid moieties of the previously reported chalcone-based proteasome inhibitor 3,5-bis(4-boronic acid benzylidene)-1-methylpiperidin-4-one and bearing a variety of amino acid substitutions on the amino group of the 4-piperidone. Our lead compound 2 (RA-1) inhibits proteasomal activity and has improved dose-dependent antiproliferative and proapoptotic properties in cervical cancer cells containing human papillomavirus. Further, it induces synergistic killing of cervical cancer cell lines when tested in combination with an FDA approved proteasome inhibitor. Exploration of the potential mechanism of proteasomal inhibition by our lead compound using in silico docking studies suggests that the carbonyl group of its oxopiperidine moiety is susceptible to nucleophilic attack by the γ-hydroxythreonine side chain within the catalytic sites of the proteasome.
[Show abstract][Hide abstract] ABSTRACT: Cervical cancer cells are addicted to the expression of the human papillomavirus (HPV) oncoproteins E6 and E7. The oncogencity of E6 is mediated in part by targeting p53 and PDZ-family tumor suppressor proteins for rapid proteasomal degradation, whereas the E7 oncoprotein acts in part by coopting histone deacetylases (HDAC)1/2. Here, we examine the hypothesis that inhibition of proteasome function and HDAC activity would synergistically and specifically trigger cervical cancer cell death by the interruption of E6 and E7 signaling.
The sensitivity and molecular responses of keratinocytes and HPV-positive and HPV-negative cervical cancer cells and xenografts to combinations of proteasome and HDAC inhibitors were tested. The expression of HDAC1/HDAC2 in situ was examined in cervical cancer, its precursors, and normal epithelium.
Cervical cancer cell lines exhibit greater sensitivity to proteasome inhibitors than do HPV-negative cervical cancers or primary human keratinocytes. Treatment of cervical cancer cells with bortezomib elevated the level of p53 but not hDlg, hScribble or hMAGI. Immunohistochemical analysis revealed elevated HDAC1/HDAC2 expression in cervical dysplasia and cervical carcinoma versus normal cervical epithelium. The combination of bortezomib and HDAC inhibitor trichostatin A or vorinostat shows synergistic killing of HPV-positive, but not HPV-negative, cervical cancer cell lines. Similarly, treatment of HeLa xenografts with the combination of bortezomib and trichostatin A retarded tumor growth significantly more effectively than either agent alone.
A combination of proteasome and HDAC inhibitors, including bortezomib and vorinostat, respectively, warrants exploration for the treatment of cervical cancer.
Clinical Cancer Research 02/2009; 15(2):570-7. DOI:10.1158/1078-0432.CCR-08-1813 · 8.72 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Elevated metabolic activity of ovarian cancer cells causes increased ubiquitin-proteasome-system (UPS) stress, resulting in their greater sensitivity to the toxic effects of proteasomal inhibition. The proteasomes and a potentially compensatory histone deacetylase 6 (HDAC6)-dependent lysosomal pathway mediate eukaryotic protein turnover. We hypothesized that up-regulation of the HDAC6-dependent lysosomal pathway occurs in response to UPS stress and proteasomal inhibition, and thus, ovarian cancer cell death can be triggered most effectively by coinhibition of both the proteasome- and HDAC6-dependent protein degradation pathways.
To address this hypothesis, we examined HDAC6 expression patterns in normal and cancerous ovarian tissues and used a novel HDAC6-specific inhibitor, NK84, to address HDAC6 function in ovarian cancer.
Abnormally high levels of HDAC6 are expressed by ovarian cancer cells in situ and in culture relative to benign epithelium and immortalized ovarian surface epithelium, respectively. Specific HDAC6 inhibition acts in synergy with the proteasome inhibitor Bortezomib (PS-341) to cause selective apoptotic cell death of ovarian cancer cells at doses that do not cause significant toxicity when used individually. Levels of UPS stress regulate the sensitivity of ovarian cancer cells to proteasome/HDAC6 inhibition. Pharmacologic inhibition of HDAC6 also reduces ovarian cancer cell spreading and migration consistent with its known function in regulating microtubule polymerization via deacetylation of alpha-tubulin.
Our results suggest the elevation of both the proteasomal and alternate HDAC6-dependent proteolytic pathways in ovarian cancer and the potential of combined inhibition of proteasome and HDAC6 as a therapy for ovarian cancer.
Clinical Cancer Research 12/2008; 14(22):7340-7. DOI:10.1158/1078-0432.CCR-08-0642 · 8.72 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Both tumor cell proliferation and metastasis are dependent on myosin II. Because UNC-45 is required to chaperone the assembly of a functional myosin II motor, we examined the expression of the general cell (GC) UNC-45 isoform in ovarian tumors. Serous carcinoma expressed elevated levels of GC UNC-45 compared with normal ovarian surface epithelium and benign cystadenoma. High-stage exhibited greater GC UNC-45 expression than low-stage serous carcinoma. Similarly, GC UNC-45 transcripts and protein levels were higher in ovarian cell lines than in immortalized ovarian surface epithelial cells. Elevation of GC UNC-45 levels by ectopic expression enhanced the rate of ovarian cancer cell proliferation, whereas siRNA knockdown of GC UNC-45 suppressed proliferation without altering myosin II levels. GC UNC-45 and myosin II were diffuse within the cytoplasm of confluent interphase cells, but both accumulated together at the cleavage furrow during cytokinesis. GC UNC-45 and myosin II also trafficked to the leading edges of ovarian cancer cells induced to move in a scratch assay. Knockdown of GC UNC-45 reduced the spreading ability of ovarian cancer cells whereas it was enhanced by GC UNC-45 overexpression. In sum, these findings implicate elevated GC UNC-45 protein expression in ovarian carcinoma proliferation and metastasis.
American Journal Of Pathology 12/2007; 171(5):1640-9. DOI:10.2353/ajpath.2007.070325 · 4.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The ubiquitin-proteasome system (UPS) mediates targeted protein degradation. Notably, the UPS determines levels of key checkpoint proteins controlling apoptosis and proliferation by controlling protein half-life. Herein, we show that ovarian carcinoma manifests an overstressed UPS by comparison with normal tissues by accumulation of ubiquitinated proteins despite elevated proteasome levels. Elevated levels of total ubiquitinated proteins and 19S and 20S proteasome subunits are evident in both low-grade and high-grade ovarian carcinoma tissues relative to benign ovarian tumors and in ovarian carcinoma cell lines relative to immortalized surface epithelium. We find that ovarian carcinoma cell lines exhibit greater sensitivity to apoptosis in response to proteasome inhibitors than immortalized ovarian surface epithelial cells. This sensitivity correlates with increased cellular proliferation rate and UPS stress rather than absolute proteasome levels. Proteasomal inhibition in vitro induces cell cycle arrest and the accumulation of p21 and p27 and triggers apoptosis via activation of caspase-3. Furthermore, treatment with the licensed proteasome inhibitor PS-341 slows the growth of ES-2 ovarian carcinoma xenograft in immunodeficient mice. In sum, elevated proliferation and metabolic rate resulting from malignant transformation of the epithelium stresses the UPS and renders ovarian carcinoma more sensitive to apoptosis in response to proteasomal inhibition.
Cancer Research 05/2006; 66(7):3754-63. DOI:10.1158/0008-5472.CAN-05-2321 · 9.33 Impact Factor