Newly developed dual topoisomerase inhibitor P8-D6 is highly active in ovarian cancer

Abstract

Background: Ovarian cancer (OvCa) constitutes a rare and highly aggressive malignancy and is one of the most lethal of all gynaecologic neoplasms. Due to chemotherapy resistance and treatment limitations because of side effects, OvCa is still not sufficiently treatable. Hence, new drugs for OvCa therapy such as P8-D6 with promising antitumour properties have a high clinical need. The benzo[c]phenanthridine P8-D6 is an effective inductor of apoptosis by acting as a dual topoisomerase I/II inhibitor.

Methods: In the present study, the effectiveness of P8-D6 on OvCa was investigated in vitro. In various OvCa cell lines and ex vivo primary cells, the apoptosis induction compared with standard therapeutic agents was determined in two-dimensional monolayers. Expanded by three-dimensional and co-culture, the P8-D6 treated cells were examined for changes in cytotoxicity, apoptosis rate and membrane integrity via scanning electron microscopy (SEM). Likewise, the effects of P8-D6 on non-cancer human ovarian surface epithelial cells and primary human hepatocytes were determined.

Results: This study shows a significant P8-D6-induced increase in apoptosis and cytotoxicity in OvCa cells which surpasses the efficacy of well-established drugs like cisplatin or the topoisomerase inhibitors etoposide and topotecan. Non-cancer cells were affected only slightly by P8-D6. Moreover, no hepatotoxic effect in in vitro studies was detected.

Conclusion: P8-D6 is a strong and rapid inductor of apoptosis and might be a novel treatment option for OvCa therapy.

Keywords: OvCa; apoptosis; chemotherapy; drug development; dual topoisomerase inhibitor; hepatotoxicity.

Figure 1.

Chemical structure and mechanism of P8-D6 action. P8-D6 acts as a dual topoisomerase inhibitor by stabilizing the cleavable Topo–DNA complex, thereby inducing apoptosis. The effectiveness and the broad activity spectrum of P8-D6 were examined for the first time in a 60-tumour cell line panel by the NCI. In the evaluation, P8-D6 reached an average ∑GI₅₀ (60) value of 49 nM in multiple tumour cell lines. For the ovarian carcinoma cells tested, this GI₅₀ average was 0.12 µM. For comparison, other active drugs are listed. ∑GI₅₀ (60): average growth inhibition 50% in 60 cancer cell lines (different cancer types), ∑GI₅₀ (OvCa): average growth inhibition 50% in OvCa cell lines.

Figure 2.

Antitumour responses in OvCa 2D monolayers. A2780 (cell line) and UF-168T (primary cells) were treated with different concentrations of P8-D6, topotecan, etoposide, cisplatin and negative control (PBS) for 48 h. Subsequently, the viability and caspase activity were determined. (a) The IC₅₀ values of each cytostatic drug were calculated by using the viability data. (b) The apoptosis is represented as relative caspase activity. (c) To compare the combinatorial apoptotic effect of topotecan (Topo I Inhibitor) and etoposide (Topo II Inhibitor) with P8-D6, a dual topoisomerase inhibitor was performed in A2780. (d) and (e) Flow cytometric analysis of pro-apoptotic effects with Annexin V-PE (An V) and 7AAD staining (n = 6). Representative flow cytometry dot plots of treated and stained A2780 cells were done. The mean distribution of viable (An V/7AAD-negative), early apoptotic (An V-positive, 7AAD-negative), late apoptotic/necrotic (An V/7AAD-positive) or necrotic (An V-negative, 7AAD-positive) tumour cells after treatment were calculated (d). (f) For primary OvCa cells (UF-168T) viability and apoptosis were measured. (g) In addition, the anti-proliferative effect after 24-h treatment was evaluated by microscopy. Scale bars, 50 µm. (h) Heat map of the IC₅₀ values using the viability of all tested OvCa cells. Data are means + SD (standard deviation) one-way ANOVA; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 3.

Antitumour properties in 3D spheroids and 2D co-culture. For 3D culture, A2780, SKOV-3, OvCar8 and BG-1 cells were maintained in ULA plates for 96 h and subsequently treated with P8-D6 (0.1, 0.5, 1 and 10 µM), topotecan (1 and 10 µM) and PBS for 48 h. (a) Every 24 h images were generated by microscopy. Scale bars, 500 µm. (b) and (c) During treatment, the cell toxicity was measured by fluorescence microscope using CellTox™ Green (24 and 48 h). Scale bars, 500 µm. The fluorescence signals for 24 and 48 h after treatment were quantified (relative fluorescence units RFU) and shown in a heat map (c). (d) After 48-h treatment, the viability and caspase activity were measured in A2780 spheroids. (e) A2780 spheroids were stained after the growth and treatment phase with PI (red), calcein-AM (green) and hoechst 33342 (blue), and measured by microscopy. Scale bars, 500 µm. (f) SEM images of A2780 and OvCar8 spheroids, which were treated with P8-D6 (1 μM) or PBS for 48 h were taken. Scale bars, 20 µm. (g) For co-culture experiments, A2780 cells were seeded in 2D monolayers on transwell inserts and fibroblasts on well bottoms. For comparison, mono-cultures were cultured and treated with P8-D6 (10 µM), etoposide (10 µM), topotecan (10 µM) and PBS in the same way. The apoptosis represented as relative caspase activity was measured in A2780 and fibroblasts. Data are means + SD (n = 3) one-way ANOVA; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 4.

Effects on non-cancer cells. (a) To examine toxic effects of therapy, human ovarian surface epithelial cells (HOSE) were treated with P8-D6, cisplatin, etoposide, topotecan and PBS for 48 h. The anti-proliferative effect after 48 h treatment was evaluated by microscopy. Scale bars, 100 µm. (b) The viability and apoptosis were measured after 48 h treatment in HOSE cells. Data are means + SD (n = 6) one-way ANOVA; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (c) To investigate the hepatotoxicity, primary human hepatocytes were treated with P8-D6, doxorubicin and PBS for 48 h. Afterwards, microscopy images were taken, and apoptosis and oxidative stress measurements were performed. Data are means + SD (n = 3) one-way ANOVA; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.