Stressing the Ubiquitin-Proteasome System without 20S Proteolytic Inhibition Selectively Kills Cervical Cancer Cells

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

Predicting response to the anti-estrogen fulvestrant in recurrent ovarian cancer

Anti-estrogen therapy appears to have efficacy in a subset of ovarian cancers, as demonstrated in multiple phase II studies. Identifying sensitive patients early in treatment may allow for targeted, low-toxicity primary therapy or prevention of recurrence. We have previously demonstrated that the likelihood of response to letrozole could be improved by patient selection based on estrogen-pathway marker expression. We sought to identify ovarian cancer biomarkers that might indicate sensitivity to fulvestrant, an estrogen receptor antagonist

Predicting response to the anti-estrogen fulvestrant in recurrent ovarian cancer

Anti-estrogen therapy appears to have efficacy in a subset of ovarian cancers, as demonstrated in multiple phase II studies. Identifying sensitive patients early in treatment may allow for targeted, low-toxicity primary therapy or prevention of recurrence. We have previously demonstrated that the likelihood of response to letrozole could be improved by patient selection based on estrogen-pathway marker expression. We sought to identify ovarian cancer biomarkers that might indicate sensitivity to fulvestrant, an estrogen receptor antagonist

EOC cell isolation by different methods of cell dissociation.

<p><b>A.</b> Mean ± standard deviation of total number of viable cells per 500 mg of tissue derived from each condition immediately after treatment (i.e. pre-adhesion cultures) and then again after 7–10 days of adhesion in tissue culture (i.e. adherent cultures). B. Percentage of cell viability for each condition expressed as percentage of pre-adhesion cultures to adherent cultures. Least-square means (adjusted for replicates within patient) of eight independent experiments and the 95% confidence intervals are presented.</p

Immunostaining of clinical specimens and freshly isolated EOC cells for EpCAM expression.

<p>A FFPE block of normal ovary, depicting positive staining for EpCAM with both the monoclonal antibodies Ber-EP4 and MOC-31 of the normal ovarian surface epithelial cells. A FFPE block of serous ovarian adenocarcinoma depicting positive staining for EpCAM with both the monoclonal antibodies Ber-EP4 and MOC-31 of the EOC cells within the tumor. Immunocytochemical staining of paraffin-embedded EOC cultures with the monoclonal antibodies Ber-EP4 and MOC-31 depicting positive staining for EpCAM. Immunocytochemical staining of paraffin-embedded fibroblast cultures with the monoclonal antibodies Ber-EP4 and MOC-31, here used as negative controls.</p

Donors characteristics.

<p>Donors characteristics.</p

Down-stream application of EOC cells for drug toxicity studies.

<p>A. Morphological changes in EOC cells either mock treated or treated with Bortezomib (2 nM) and Vorinostat (6 µM) alone and in combination for 18 hours. B. Representative examples of lysate of EOC cells from donors 1 and 8 (<i>top and middle panel</i>) or ES-2 human ovarian cancer cell line (<i>bottom panel</i>) either mock treated or treated with Bortezomib (2 nM) and Vorinostat (6 µM) or Bortezomib (6 nM) and Vorinostat (3 µM) respectively, alone and in combination for 18 hours and immunoblotted with an antibody recognizing the full-length and the cleaved forms of PARP. Equal protein loading was verified by using an antibody directed against β-actin. * = a specific band. C. Increase in the cleaved-PARP species in the mock versus treated EOC cells expressed as cleaved full length/cleaved PARP ratio.</p

Morphologic characteristics of EOC cells.

<p>(a) Representative examples of primary EOC cells isolated from cancer specimen after two days in culture (arrow indicates erythrocytes); (b) Swirl-like clusters of EOC cells after 3–4 days in culture (arrow indicates the growing clusters); (c) A colony of EOC cells spreading on the tissue culture plastic after 7–8 days in culture; (d) A confluent monolayer of EOC cells depicting typical epithelial cobblestone morphology after 13–14 days in culture; and (e) A confluent monolayer of fibroblasts here used as negative control.</p

Down-stream application of EOC cells for anchorage independent growth and transduction efficiency.

<p>(a) EOC cells from donor 8 forming spheroids-like structures on agarose; (b) NIH:OVCAR5 human ovarian cancer cell line also formed spheroids (positive control); (c) EOC cells from donor 8 transfected with the GFP expressing lentivirus pEF-GFP-SIN; and (d) phase contrast of the same field.</p