tolfenamic-acid and Ovarian-Neoplasms

The expression of specificity protein 1 (Sp1) and survivin was evaluated in clinical specimens of epithelial ovarian cancer (EOC) patients. When compared to normal tissue, EOC samples showed high expression of Sp1 and survivin using qPCR (Sp1: ∼2-fold; survivin: ∼5-fold) and Western blot (Sp1: >2.6-fold; survivin: >100-fold). The Sp1 inhibitor, and anti-cancer small molecule, tolfenamic acid (TA), was tested to enhance the response of Cisplatin (Cis) in EOC cell lines. Cell viability (CellTiter-Glo), combination index (CalcuSyn software), apoptosis (Annexin-V staining), cell cycle analyses (flow cytometry), and reactive oxygen species (flow cytometry) were determined. Cell migration and invasion was assessed using matrigel coated transwell chambers. Agilent Technologies proteomics analysis identified potential signaling pathways involved. The combination of TA (50 μM) and Cis (5 μM) synergistically increased the growth inhibition in ES2 (∼80 %, p < 0.001) and OVCAR-3 (60 %, p < 0.001) cells. TA or TA + Cis treatment in ES2 cells caused cell cycle arrest in G1 Phase (TA) or S-Phase (TA + Cis) and unregulated reactive oxygen species. Invasion and migration was decreased in ES2 cells. Global proteomic profiling showed modulation of proteins associated with oxidative phosphorylation, apoptosis, electron transport chain, DNA damage, and cell cycle proteins. These results demonstrate an association of Sp1 and survivin in EOC and confirm targeting these candidates with TA potentially sensitizes EOC cells to cisplatin.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents; Apoptosis; Biomarkers, Tumor; Blotting, Western; Carcinoma, Ovarian Epithelial; Cell Cycle; Cell Movement; Cell Proliferation; Cisplatin; Drug Therapy, Combination; Female; Humans; Inhibitor of Apoptosis Proteins; Neoplasms, Glandular and Epithelial; ortho-Aminobenzoates; Ovarian Neoplasms; Proteomics; Reactive Oxygen Species; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sp1 Transcription Factor; Survivin; Tumor Cells, Cultured

The aberrant expression of hepatocyte growth factor and its receptor c-Met are associated with aggressive disease and poor prognosis in a variety of human malignancies including ovarian cancer (OC). Specificity protein (Sp) transcription factors have high relevance in the signaling cascade associated with c-Met activation. Tolfenamic acid (TA), a NSAID, is known to induce the degradation of Sp proteins, which have been negatively associated with survival in some cancer patients. Our aim was to examine the anti-OC activity of TA using in vitro and in vivo models and asses the inhibitory effects of this novel compound.. We developed OC sub-cell lines (AF1-3) derived from the original SKOV3 that are more resistant to IFNα-2b and more tumorigenic in nude mice than the original cells. We tested the anti-cancer activity of TA using ovarian cancer cells, SKOV3-AF2 and ES-2. The cells were treated with DMSO (vehicle) or TA (25/50/100 μM) and cell viability was measured at 24, 48, and 72 h. Cell lysates were prepared following 48 h treatment (50μM) and evaluated the expression of Sp proteins (Sp1/Sp3/Sp4), c-Met, survivin, Bcl2, and cleaved polyADP-ribose polymerase (c-PARP) through Western blot analysis. Caspase-3 activity was assessed with Caspase-Glo kit. Cell cycle distribution and apoptosis were analyzed using BD FACSCalibur flow cytometer. For in vivo studies mice were subcutaneously injected with ES-2 cells and treated with vehicle or TA (50 mg/kg/thrice weekly).. TA significantly inhibited the growth of SKOV3 (AF1-3) and ES-2 cells. The expression of Sp proteins and c-Met was significantly decreased suggesting that TA could be targeting c-Met through degradation of Sp proteins. TA greatly increased the apoptotic fraction (Annexin V positive), c-PARP expression and caspase-3 activity. TA significantly decreased Bcl2 expression and induced G0/G1 cell cycle arrest. In vivo studies revealed that TA significantly inhibited tumor weight and volume in mice. These results show that TA has a profound inhibitory effect on tumor growth in mice, reduces OC cells proliferation, induces apoptosis, cell cycle arrest and Sp proteins degradation. TA also inhabited the expression of survivin that is associated with radiation resistance and suggests that apart from its tumor suppressant effects, TA can also enhance the tumor response to radiotherapy.. These data clearly show that TA effectively inhibits OC cell growth in vitro and in vivo. This study represents potential role(s) of TA that suppresses OC cell growth, and may enhance tumor response to radiotherapy, and have implications in OC treatment.

Topics: Animals; Apoptosis; Cell Cycle; Cell Growth Processes; Cell Survival; Female; Humans; Inhibitor of Apoptosis Proteins; Mice; Mice, Nude; ortho-Aminobenzoates; Ovarian Neoplasms; Proto-Oncogene Proteins c-met; Sp Transcription Factors; Survivin; Xenograft Model Antitumor Assays