Evaluation of the cytotoxicity of the Bithionol-paclitaxel combination in a panel of human ovarian cancer cell lines.

Previously, Bithionol (BT) was shown to enhance the chemosensitivity of ovarian cancer cell lines to cisplatin treatment. In the present study, we focused on the anti-tumor potential of the BT-paclitaxel combination when added to a panel of ovarian cancer cell lines. This in vitro study aimed to 1) determine the optimum schedule for combination of BT and paclitaxel and 2) assess the nature and mechanism(s) underlying BT-paclitaxel interactions. The cytotoxic effects of both drugs either alone or in combination were assessed by presto-blue cell viability assay using six human ovarian cancer cell lines. Inhibitory concentrations to achieve 50% cell death (IC50) were determined for BT and paclitaxel in each cell line. Changes in levels of cleaved PARP, XIAP, bcl-2, bcl-xL, p21 and p27 were determined via immunoblot. Luminescent and colorimetric assays were used to determine caspases 3/7 and autotaxin (ATX) activity. Cellular reactive oxygen species (ROS) were measured by flow cytometry. Our results show that the efficacy of the BT-paclitaxel combination depends upon the concentrations and sequence of addition of paclitaxel and BT. Pretreatment with BT followed by paclitaxel resulted in antagonistic interactions whereas synergistic interactions were observed when both drugs were added simultaneously or when cells were pretreated with paclitaxel followed by BT. Synergistic interactions between BT and paclitaxel were attributed to increased ROS generation and enhanced apoptosis. Decreased expression of pro-survival factors (XIAP, bcl-2, bcl-xL) and increased expression of pro-apoptotic factors (caspases 3/7, PARP cleavage) was observed. Additionally, increased expression of key cell cycle regulators p21 and p27 was observed. These results show that BT and paclitaxel interacted synergistically at most drug ratios which, however, was highly dependent on the sequence of the addition of drugs. Our results suggest that BT-paclitaxel combination therapy may be effective in sensitizing ovarian cancer cells to paclitaxel treatment, thus mitigating some of the toxic effects associated with high doses of paclitaxel

Assessment of acyl-CoA cholesterol acyltransferase (ACAT-1) role in ovarian cancer progression-An in vitro study.

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

Apoptotic effects of BT-paclitaxel combination on isogenic pairs of ovarian cancer cell lines.

<p><b>(A and C)</b> Representative images of Hoechst 33258 staining (for morphological, qualitative assessment of apoptosis) of A2780 and A2780-CDDP <b>(A)</b> or IGROV and IGROV-1-CDDP (<b>C)</b> cells treated with BT and/or paclitaxel as indicated. Percent of apoptosis in terms of DNA fragmentation (quantified via TUNEL assay) for A2780 and A2780-CDDP <b>(B)</b> or IGROV and 1/IGROV-1-CDDP <b>(D)</b> cells treated with BT or paclitaxel alone or in combination. The fragmentation observed in vehicle treated cells (control) is considered as zero percent against which treated cells were compared. Experiments were performed in duplicate. Data were expressed as means ± SD of duplicate experiments. Comparisons between paclitaxel alone treated and combination treated for each cell line were performed using Student’s t-test. Asterisks (*) indicate p < 0.05.</p

Assessment of apoptosis induced by BT-paclitaxel combination on ovarian cancer cell lines.

<p>The effect of BT-paclitaxel combination on caspase 3/7 activity was measured in <b>(A)</b> A2780 and A2780-CDDP or <b>(B)</b> IGROV-1 and IGROV-1-CDDP. Vehicle-treated cells were considered as control against which treated cells were compared. Data were expressed as means ± SD of triplicate experiments. Comparisons between paclitaxel alone-treated and combination-treated for each cell line were performed using Student’s t-test. Asterisks (*) indicate p < 0.05. The effects of BT-paclitaxel combination on pro-apoptotic (cPARP), anti-apoptotic (XIAP, bcl-2, bcl-xL) and cell cycle regulatory markers were assessed on A2780 and A2780-CDDP (<b>C)</b> or IGROV-1 and IGROV-1-CDDP <b>(D)</b>. Analysis of the expression of proteins in the lysates of treated and untreated (control) cells was carried out by SDS-PAGE and western blot analysis. The blots were probed with the respective primary antibodies. As an internal standard for equal loading, blots were probed with an anti-<i>β</i>-actin antibody. All blots were quantified using Odyssey software and data presented in the supporting information section, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185111#pone.0185111.s003" target="_blank">S2 Fig</a>.</p

Assessment of intracellular ROS and antioxidant effect on ovarian cancer cells treated with BT-paclitaxel combination.

<p>Flow cytometry detection of intracellular ROS in A2780 and A2780-CDDP <b>(A)</b> or IGROV-1 and IGROV-1-CDDP <b>(B)</b> cells treated with BT or paclitaxel alone or in combination. Data were expressed as mean ± SD of triplicate experiments. Comparisons between paclitaxel alone treated and combination treated for each cell line were performed using Student’s t-test. Asterisks (*) indicate p < 0.05. <b>(C)</b> and <b>(D)</b> show the effect of the antioxidant ascorbic acid on the viability (via PrestoBlue) of A2780 and A2780-CDDP (<b>C)</b> or IGROV-1 and IGROV-1-CDDP <b>(D)</b> cells treated with BT or paclitaxel alone or in combination. Control (untreated) cells were considered as 100% viable against which treated cells were compared. Data were expressed as means ± SD of triplicate experiments. Comparisons between BT-paclitaxel-treated in presence of AA vs. combination-treated in the absence of AA for each cell line were performed using Student’s t-test. Asterisks (*) indicate p < 0.05.</p

Cytotoxic potential of BT-paclitaxel combination on the isogenic pair of ovarian cancer cell lines IGROV-1 (cisplatin-sensitive) and IGROV-1-CDDP (cisplatin-resistant).

<p><i>C</i>ombination index (CI) values were calculated and represented as heat maps where a drug combination is synergistic (green color) if CI < 0.9; additive (yellow color) if CI is between 0.9 and 1.0; and antagonistic (red color) if CI > 1.0. <b>(A)</b> and <b>(C)</b> show CI values for IGROV-1 and IGROV-1-CDDP, respectively. Dose response curves for IGROV-1 <b>(B)</b> and IGROV-1-CDDP <b>(D)</b> when treated with paclitaxel alone or BT/paclitaxel combination (simultaneous addition). All experiments were performed in triplicate.</p

Cytotoxic potential of BT-paclitaxel combination on the isogenic pair of ovarian cancer cell lines A2780 (cisplatin-sensitive) and A2780-CDDP (cisplatin-resistant).

<p>Combination index (CI) values were calculated based on the percentage viability of cells treated with combinations of BT and paclitaxel. The nature of BT-paclitaxel interactions is represented in heat maps where synergism is shown in green (if CI < 0.9); additive effects are shown in yellow color (if CI is between 0.9 and 1.0); and antagonism is shown in red (if CI > 1.0). Combination index values for A2780 and A2780-CDDP are shown in <b>(A)</b> and <b>(C),</b> respectively. Dose response curves for A2780 <b>(B)</b> and A2780-CDDP <b>(D),</b> respectively when treated with paclitaxel alone or BT-paclitaxel combination (simultaneous addition). All experiments were performed in triplicate.</p