Abstract
Abnormal accumulation of acyl-CoA cholesterol acyltransferase-1 (ACAT-1) mediated cholesterol ester has been shown to contribute to cancer progression in various cancers including leukemia, glioma, breast, pancreatic and prostate cancers. However, the significance of ACAT-1 and cholesterol esters (CE) is relatively understudied in ovarian cancer. In this in vitro study, we assessed the expression and contribution of ACAT-1 in ovarian cancer progression. We observed a significant increase in the expression of ACAT-1 and CE levels in a panel of ovarian cancer cell lines (OC-314, SKOV-3 and IGROV-1) compared to primary ovarian epithelial cells (normal controls). To confirm the tumor promoting capacity of ACAT-1, we inhibited ACAT-1 expression and activity by treating our cell lines with an ACAT inhibitor, avasimibe, or by stable transfection with ACAT-1 specific short hairpin RNA (shRNA). We observed significant suppression of cell proliferation, migration and invasion in ACAT-1 knockdown ovarian cancer cell lines compared to their respective controls (cell lines transfected with scrambled shRNA). ACAT-1 inhibition enhanced apoptosis with a concurrent increase in caspases 3/7 activity and decreased mitochondrial membrane potential. Increased generation of reactive oxygen species (ROS) coupled with increased expression of p53 may be the mechanism(s) underlying pro-apoptotic action of ACAT-1 inhibition. Additionally, ACAT-1 inhibited ovarian cancer cell lines displayed enhanced chemosensitivity to cisplatin treatment. These results suggest ACAT-1 may be a potential new target for the treatment of ovarian cancer.
Highlights
Using Quantitative real time-PCR (qRT-PCR), we found significant expression of acyl-CoA cholesterol acyltransferase (ACAT)-1 mRNA in a panel of ovarian cancer cell lines compared to primary ovarian epithelial cells, H-6036 (Fig 1A)
ACAT-1 role in ovarian cancer progression threshold cycle (Ct) values were normalized to the housekeeping gene 18srRNA to quantify ACAT-1 mRNA (Comparative Ct method)
GAPDH is used as the loading control. (D) Immunocytochemical staining for ACAT-1 in various ovarian cancer cell lines using primary antibody against ACAT-1 with FITC labeled secondary antibody and DAPI counterstain
Summary
Human primary ovarian epithelial cells (H-6036) were obtained from Cell Biologics, (Chicago, IL, USA). Human ovarian carcinoma cell lines, OC-314 and SKOV-3 were obtained from Dr McAsey’s laboratory (Department of Obstetrics & Gynecology, SIU School of Medicine, Springfield, IL). Isogenic ovarian cancer cell line pairs, e.g., A2780 / A2780-CDDP and IGROV-1 / IGROV-1CDDP were obtained from Dr Brodsky (Brown University, Providence, RI). As previously reported [30], all cell lines were maintained in DMEM media (Sigma) supplemented with 10% heat inactivated FBS (Hyclone), 10 mM HEPES (Mediatech), 4 mM L-. All cell lines were cultured at 37 ̊C in a humidified atmosphere with 5% CO2. SKOV-3, IGROV-1 and OC314 cell lines were authenticated by the ATCC using STR profiling technique. Avasimibe used in the experiments was purchased from Selleckchem, TX, USA
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