Ormeloxifene efficiently inhibits ovarian cancer growth

Abstract

Ovarian cancer continues to be a leading cause of cancer related deaths for women. Anticancer agents effective against chemo-resistant cells are greatly needed for ovarian cancer treatment. Repurposing drugs currently in human use is an attractive strategy for developing novel cancer treatments with expedited translation into clinical trials. Therefore, we examined whether ormeloxifene (ORM), a non-steroidal Selective Estrogen Receptor Modulator (SERM) currently used for contraception, is therapeutically effective at inhibiting ovarian cancer growth. We report that ORM treatment inhibits cell growth and induces apoptosis in ovarian cancer cell lines, including cell lines resistant to cisplatin. Furthermore, ORM treatment decreases Akt phosphorylation, increases p53 phosphorylation, and modulates the expression and localization patterns of p27, cyclin E, cyclin D1, and CDK2. In a pre-clinical xenograft mouse ORM treatment significantly reduces tumorigenesis and metastasis. These results indicate that ORM effectively inhibits the growth of cisplatin resistant ovarian cancer cells. ORM is currently in human use and has an established record of patient safety. Our encouraging in vitro and pre-clinical in vivo findings indicate that ORM is a promising candidate for the treatment of ovarian cancer.

Keywords: Apoptosis; Cisplatin resistance; Novel therapies; Ormeloxifene; Ovarian cancer; Xenograft mice.

Fig. 1. Ormeloxifene treatment inhibits growth of chemo-sensitive (A2780) and chemo-resistant (A2780-CP and SKOV3) ovarian cancer cell lines

A) Proliferation was determined with an MTS assay 48 hours after ORM addition and normalized to control wells treated with appropriate amounts of vehicle (ethanol, set at 100%, labeled as 0 ORM). Columns: mean, Bars: SE, n=3. *indicates p value: <0.05. B) A colony forming assay was conducted to determine the long term effect of ORM treatment on the clonogenic potential of ovarian cancer cells. Colonies were counted and expressed as a percentage of the number of colonies in the vehicle control (ethanol, set at 100%, labeled as 0 ORM). Columns: mean, Bars: SE, n=3. *indicates p<0.05. Representative plates are shown for A2780-CP cells.

Fig. 2. ORM treatment causes apoptosis through decreased mitochondrial membrane potential (ΔΨm) and activated apoptosis associated proteins

A–B) ORM treatment leads to apoptosis. Ovarian cancer cells were treated with ORM for 48 hours and processed for staining with the APO-BrdU™ TUNEL Assay Kit. DNA fragmentation was analyzed by flow cytometry. A) Percent TUNEL positive cells are shown for each cell line tested. *indicates p value: <0.05 compared to vehicle control. B) Representative FSC/FL-1 plots are shown for SKOV-3 cells. Data are representative of one of three similar experiments. C) ORM treatment leads to cleavage of caspases 3 and 9 and PARP. Cells were treated with indicated concentrations of ORM for 48 h, protein lysates were collected, 20µg of protein was resolved by SDS-PAGE and immunoblotted with primary antibodies as indicated. β-actin was used as an internal loading control. Western blots shown are representative from one of two similar experiments. D and E) ORM decreases mitochondrial potential in a dose and time dependent response. Cells were treated with ORM as indicated and live cells were incubated in TMRE and analyzed by flow cytometry. D) Graphs show the change in mean fluorescence values normalized to vehicle control (ethanol, labeled as 0 ORM)). *indicates p value: <0.05. E) A representative histogram of A2780-CP after 18h of ORM treatment is shown. Data are representative of one of three similar experiments.

Fig. 3. ORM treatment induces changes in cell cycle related proteins in ovarian cancer cells

A–C) ORM treatment alters cell cycle distribution. After 48h of ORM treatment, adherent and floating cells were collected, fixed with 70% ethanol, stained with Telford Reagent containing propidium iodide, incubated overnight at 4°C, and analyzed by flow cytometry for cell cycle analysis. A) Representative histograms are shown for A2780-CP cells. Graphs show the percent of cells in B) G₀G₁ and C) S phase. *indicates p value: <0.05. D) ORM treatment alters proteins associated with cell cycle progression. Cells were treated with indicated concentrations of ORM for 48 h, protein lysates were collected, separated on SDS-PAGE, and immunoblotted for proteins as indicated. β-actin was used as an internal control. Western blots shown are representative from one of two similar experiments.

Fig. 4. ORM treatment decreases nuclear localization of cell cycle related proteins

Ovarian cancer cells, A2780, A2780-CP and SKOV-3 cells were treated with 10 µM ORM as indicated for 18h, fixed, and stained for the detection of p27, cyclin D1, cyclin E, and CDK2 (green staining) with DAPI counter stain (nuclear marker, blue). Confocal fluorescence microscopy shows that ORM decreases nuclear localization of cell cycle related proteins. (Image on the left is green only, right hand image is merged with the DAPI nuclear counter stain).

Fig. 5. ORM inhibits ovarian cancer tumorigenesis in mice

A) Schedule of ormeloxifene treatment in ovarian cancer xenograft mouse model. Mice were injected with 50 or 100 µg ORM once a week for 5 weeks as indicated (D=days). Ethanol (EtOH) was the vehicle control. B) Images of mice from each group injected with ORM or ethanol control. Graphs show the average number of tumors (top) and metastases (bottom) observed in mice treated with ORM or ethanol (vehicle control). Data expressed as mean ± SE of 6 mice in each group. *indicates p value: <0.001.