Abstract
Objective: The limitations of therapy in ovarian cancer patients are chemoresistance and side effects. Phenoxodiol (Ph), a synthetic analogue of genistein, induces apoptosis in ovarian cancer cells by specifically removing the blockers of apoptosis. In the present in vivo and in vitro study, we demonstrated Ph induces sensitivity to chemoresistant ovarian cancer cells to low levels of chemotherapy. Methods: The in vitro studies were done using ovarian cancer cells isolated from ascites using an immunomagnetic assay and an established ovarian cancer cell line (CP70). Cell viability was determined using CellTiter?. Apoptosis was evaluated using Hoechst 33342 dye. The in vivo effect was tested by injecting A2780 subcutaneously into nude mice. Animals received daily oral administration of Ph, 10 or 20 mg/kg for 8 days alone or in combination with cisplatin 0.5 mg/kg. After 8 days the animals were sacrificed and the tumor volume was measured. Results: Ph treatment for 48 hours (h) induces 60-80% decrease in cell viability in carboplatin and paclitaxel resistant cells. Pre-treatment with Ph alone for 2 h decreased cell viability by 20%. Furthermore, pre-treatment (2 h) with Ph in chemoresistant cells followed by carboplatin or paclitaxel for 48 h resulted in a 30% and 50% significant decrease in cell viability, respectively. Hoechst stain confirmed the presence of apoptosis in the treated cells. In vivo, cisplatin (0.5 mg/kg) had no effect on tumor size while the combination of Ph (10mg/kg) and cisplatin (0.5 mg/kg) reduced tumor mass by 75% (p=0.05). Conclusion: This data suggests that Ph may restore the sensitivity to standard chemotherapies in resistant ovarian cancer cells by interacting with apoptotic blockers. A clinical trial is being conducted to assess the efficacy of Ph as a monotherapy in patients with recurrent ovarian carcinoma. A future clinical trial will need to be conducted to confirm the findings demonstrated in these in vitro and in vivo experiments and to assess the efficacy of a combination therapy regimen that includes Ph.

Introduction
Ovarian cancer is the fourth leading cause of cancer death and the most lethal of the gynecologic malignancies.

New therapies have led to improvements in the five-year survival, yet there has been no improvement in the overall survival.

The limitations of therapy in ovarian cancer patients are chemoresistance and side effects.

Phenoxodiol, an isoflavone derivative, induces apoptosis in ovarian cancer cells by specifically removing the blockers of apoptosis .

Objective
To restore chemosensitivity to previously resistant ovarian cancer cells to low levels of chemotherapy by pre-treatment with Phenoxodiol.

Methods
In vitro studies were performed using primary epithelial ovarian cancer cells isolated from ascites of ovarian cancer patients and the established ovarian cancer cell lines CP70 and A2780.

The IC50 was determined using the CellTiter© assay.

Caspase activation was determined by Western blot analysis.

In vivo studies were performed using a nude mice animal model injected with A2780 subcutaneously into the bilateral flanks. Eight animals in each treatment arm received daily oral administration of Phenoxodiol, 10 or 20 mg/kg for 8 days alone or in combination with intraperitoneal cisplatin 0.5 mg/kg. After 8 days the animals were sacrificed and the tumor volume was measured.

Results
The IC50 for carboplatin ranged from 60 µg/ml to greater than 100 µg/ml (figure 1) The IC50 for paclitaxel in the paclitaxel resistant cell line, R182, was greater than 2uM (figure 2)

Pre-treatment with Phenoxodiol (10 µg/ml) for two hours significantly reduced the IC50 for carboplatin (0.5 µg/ml +/- 0.5) and paclitaxel (0.05 µM) (figures 3 and 4).

Western blot analysis demonstrated that resistant ovarian cancer cells expressed high levels of active XIAP. Additionally, we did not detect the active form of caspase 3 in chemoresistant cells. Caspase 3 activation was observed in the chemoresistant cells only after pre-treatment with Phenoxodiol (figure 5). In vivo, cisplatin (0.5 mg/kg) had no effect on tumor size while the combination of Phenoxodiol (10mg/kg) and cisplatin (0.5 mg/kg) reduced tumor mass by 75% (p<0.05) (figure 6).

Figure 1

Figure 2

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Figure 5

Figure 6

Conclusions
Combining chemotherapy with Phenoxodiol as a treatment for epithelial ovarian cancer cells significantly reduced the IC50 of standard chemotherapies, thereby increasing sensitivity of the cancer cells to chemotoxic agents.

A clinical trial is currently being conducted to assess the efficacy of Phenoxodiol as a monotherapy while a future clinical trial will be performed to evaluate the efficacy of Phenoxodiol in a combinational regimen.