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

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.


Background
Ovarian cancer (OvCa) is the fifth leading cause of cancer deaths among women and the most lethal gynaecological malignancy in the developed world. 1 The frequent diagnosis in advanced stages and insufficient treatment options due to chemotherapy resistance and side effects lead to a poor prognosis. First line therapy usually consists of surgical cytoreduction followed by platinum/taxane-based combination chemotherapy, but most patients relapse with drug-resistance in the course of the disease. 2 Previous studies demonstrated a benefit in second line therapy with topoisomerase inhibitors like liposomal doxorubicin or topotecan. However, response duration was commonly short. 3 Consequently, an important aim is to reduce mortality by improved new therapeutic options.
P8-D6 acts as a dual topoisomerase poison by stabilizing the covalent Topo-DNA-intermediate of both topoisomerase (Topo) enzymes I and II. 4 Topoisomerases regulate torsional stress in DNA to enable essential genome functions (e.g. transcription, replication or recombination). Topo I Newly developed dual topoisomerase inhibitor P8-D6 is highly active in ovarian cancer causes single strand breaks while Topo II with its isoforms α and β is responsible for the doublestrand break ( Figure 1). 5,6 Such topoisomerase poisoning leads to cell death by inducing apoptosis. The aza-analogous benzo[c]phenanthridine P8-D6 was synthesized in an optimized four-step process with advantageous physicochemical and cytotoxic properties. 7 In the National Cancer Institute (NCI)-60 DTP Human Tumor Cell Line screening 49 nM of P8-D6 results in an average growth inhibition of 50% (GI 50 ). 7,8 The result for OvCa cell lines were 0.12 µM compared with cisplatin with 15.25 µM or topotecan with 0.23 µM, respectively.
Cell-based assays are an important pillar in drug development. In addition to traditional twodimensional (2D) monolayer, co-culture and three-dimensional (3D) cell culture have recently gained importance because of greater comparability with in vivo set ups. 9 Therefore, efficacy studies of P8-D6 were performed on different OvCa cell lines and patient-derived primary cells, compared with single drugs and combinational drug therapy. [10][11][12] Preclinical cancer drug development addressed several topics, including target achievement, induction of apoptosis in cancer cells and toxicity in normal cells.

Materials
P8-D6 was synthesized as recently described 7 and solved in phosphate-buffered saline (PBS). Topotecan, etoposide, cisplatin and doxorubicin were obtained from the UKSH dispensary.
In vitro experiments Cell preparation and culture. Human OvCa cell lines A2780, BG-1, Igrov-1, OvCar8, SKOV-3 and fibroblasts Detroit 551 were maintained in RPMI 1640 supplemented with 10% fetal bovine serum 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 ∑GI 50 (60) value of 49 nM in multiple tumour cell lines. 7 For the ovarian carcinoma cells tested, this GI 50 average was 0.12 µM. For comparison, other active drugs are listed. ∑GI 50 (60): average growth inhibition 50% in 60 cancer cell lines (different cancer types), ∑GI 50 (OvCa): average growth inhibition 50% in OvCa cell lines.
Informed consent was obtained from all donors, in agreement with the approval from the Institutional Review Board of the UKSH, Campus Kiel (AZ: D578/20) and the Uppsala Regional Ethical Review Board (Ethical Approval 2009/028).
Hepatotoxicity. Oxidative stress of hepatocytes caused by 48 h treatment was analysed via dihydroethidium fluorescence. The cell culture protocol was previously described. 15 Apoptosis was analysed using ApoLive-Glo™ Multiplex Assay.
Statistical analysis. Statistical tests were performed using GraphPad Prism9 (GraphPad). Gaussian distribution was tested by Shapiro-Wilk normality test. Data of multiple groups were checked with one-way analysis of variance (ANOVA) for statistical significance. Statistically significant differences were assumed at p values < 0.05 (*) according to Tukey´s multiple comparison and Dunn's method.

Results
Previous studies showed that P8-D6 functions as dual topoisomerase inhibitor. 4,7 However, the effectiveness is highly dependent on reaching its nuclear target structure -topoisomerase I/II. In addition to colon cancer cells, 4 we checked the location of P8-D6 and Topo I and II expression in OvCa. Using fluorescent microscopy, P8-D6 was identified in the nucleus (Additional file 1A and B in the Supplemental material). In addition, western blot provided evidence that all cells also express sufficient Topo I and Topo II (Additional file 1C in the Supplemental material).

P8-D6 is highly effective in OvCa 2D monolayers
The main aim of this study was to prove efficacy of P8-D6 in OvCa. Therefore, OvCa cell lines (A2780, Igrov-1, BG-1, OvCar8, SKOV-3, SKOV-3luc) were treated with P8-D6, and compared with topotecan, etoposide and cisplatin. Initially, the viability of OvCa cells after 48 h treatment was measured using an enzymatic assay and IC 50 -values were determined. P8-D6 exhibits a four times lower IC 50 -value and is, therefore, significantly more effective than the standard chemotherapeutics (Figure 2 A significantly higher increase of apoptosis after 48 h treatment with P8-D6 compared with its standard therapeutic drugs could be observed in all tested OvCa cell lines by using ApoLive-Glo™ Multiplex Assay (Figure 2(b), Additional file 2B-F in the Supplemental material) and flow cytometric analyses (Figure 2(d) and (e); Additional file 3B in the Supplemental material).
Since P8-D6 is a dual topoisomerase inhibitor, but the reference substances primarily inhibit one of the enzymes only, a combination of a Topo I (topotecan) and a Topo II (etoposide) inhibitor was analysed. Compared with this combination, P8-D6 shows a significantly higher rate of apoptosis (Figure 2(c); Additional file 3A in the Supplemental material). To validate the pronounced induction of apoptosis and anti-proliferative effect of P8-D6 in primary cells, we used ex vivo patient-derived cells from tumour tissue and ascites in a translational aspect. A significantly higher rate of anti-proliferative and apoptotic effect was observed in primary cells by P8-D6 compared with comparative substances (Figure 2(f) and (g); Additional file 2G and H in the Supplemental material). Altogether, we generated results of a plurality of different cells (established and primary cells) to emphasize the durable and robust effect of P8-D6 (Figure 2(h)).

P8-D6 induce strong effects in 3D target tumour and co-culture model
The 3D cell cultures are regarded to bridge the gap from 2D to in vivo models, since cell-cell interaction is considerable for the efficacy of a substance. The 3D spheroids mimic the physiological behaviour of solid tumours more closely. 10,11 Spheroids were generated in ULA plates for 96 h and subsequently treated with P8-D6 or topotecan. The OvCa spheroids (A2780, SKOV-3, BG-1, OvCar8) showed a decrease in growth behaviour and stability after P8-D6 treatment (Figure 3(a); Additional file 4A-C in the Supplemental material). Cell toxicity was increased in all spheroids by P8-D6 compared with topotecan and PBS (Figure 3   In addition, it was investigated whether fibroblast-A2780 co-culture could mediate changes in cancer cell responses to anti-cancer drugs by affecting cell-cell interaction (Figure 3(g)).
Co-culture promotes the apoptosis induction significantly for P8-D6 treated cancer cells compared with mono-culture, while the co-culture treated cancer cells with etoposide and topotecan only exhibited minimal activity differences.

P8-D6 only slightly affects non-cancer cells
Since side effects often occur as a result of tumour therapy, it is important to determine the toxicity of P8-D6 in preclinical setting on non-cancerous cells such as HOSE. HOSE were treated for 48 h and the anti-proliferative effect of P8-D6 is only slightly increased compared with the reference (Figure 4(a)). While 1 µM P8-D6 is highly effective on cancer cells, no significant increase of apoptosis was measured compared with PBS control in HOSE cells. The application of 10 µM P8-D6 shows a similar value as positive controls (Figure 4(b)).
Hepatotoxicity is a common and serious side effect in chemotherapy. By measuring oxidative stress and induction of apoptosis after 48-h  (Figure 4(c)). P8-D6 proved to be cytotoxic against OvCa cells without inducing cell death in hepatocytes.

Discussion
Since OvCa is one of the world's deadliest gynaecological malignancies, there is a high clinical need for the development of new, effective and well-tolerated therapy options with suitable physiochemical properties. 1 P8-D6 was investigated as a novel dual topoisomerase inhibitor in OvCa cell line and ex vivo patient-derived primary cells in 2D, 3D and co-culture, and was proven to be overall significantly more effective than standard therapeutics and negative control.
Side effects due to chemotherapy have significant impacts on therapy. However, P8-D6 shows only limited toxic effects on normal cells measured by oxidative stress and induces apoptosis. The significantly reduced influences on the normal cells could be explained by the reduced ability of these cells to divide, but these causes have to be investigated furthermore. If efficacy and toxicity studies are related to clinical therapy, P8-D6 induces significantly higher (>five-fold) apoptosis rates in OvCa than current standards. Thus, it could potentially be used in lower doses than the standard therapeutic agents to achieve the same effect.
Due to the dual topoisomerase inhibition of P8-D6, the question arises whether a combination therapy of two established mono-Topo I/II inhibitors reach the antitumour potency of P8-D6 in OvCa. Previous studies compared the combination of Topo I with Topo II inhibitor versus monotherapy and showed heterogeneous results. [18][19][20][21] This study showed significantly higher induction of apoptosis by P8-D6 when compared with the additive effect of Topo I and Topo II inhibitor combination. So far, the combination of topotecan and etoposide shows no clinical benefit 22 and is not considered as a clinical standard, whereas the monotherapy of topotecan is proposed to treat in second line therapy. 23,24 A major advantage of a dual topoisomerase inhibitor is the reduced development of resistance through inhibition of both topoisomerases. The inhibition of only one enzyme causes a compensatory upregulation of the other. 21,25 The effectiveness of dual topo-inhibitors on solid tumours has been shown previously. 26,27 In clinical trials, however, these drugs showed intolerable side effects (e.g. intoplicine, TA-103, batracylin), such as hepatotoxicity and severe neutropenia. [27][28][29][30][31][32][33] Because of this increased hepatotoxicity with dual topoisomerase inhibitors, 34 the hepatotoxicity of P8-D6 was determined. Importantly, this study shows that P8-D6 has no relevant effect concerning oxidative stress and apoptosis in human hepatocytes. The mechanism of action is the key benefit and novel about these drugs by affecting both topoisomerases. However, it is a validated target since single Topo inhibitors are already standards in cancer therapy. 23,24,35 The current therapy for platinum-resistant/refractory OvCa consists mainly of a monotherapy with paclitaxel, gemcitabine or PLD (pegylated liposomal doxorubicin), but cancer control rate is limited. 36,37 In addition, topotecan is another approved treatment option for platinum-resistant/ refractory OvCa therapy. 38,39 Hence, some novel drugs are in clinical trials like the tyrosine kinase inhibitor rivoceranib. 40 The antibody drug conjugate (ADC) Mirvetuximab soravtansine (MIRV) comprises a Folate receptor alpha-binding antibody, cleavable linker and the tubulin-targeting toxin DM4. A Phase III study evaluated the safety and efficacy of MIRV compared with chemotherapy in patients with platinum-resistant OvCa. 41 A combination of P8-D6 with a tyrosyl-DNA phosphodiesterase inhibitor or a Poly (ADPribose) polymerase (PARP) inhibitor could merit additional consideration. These enzymes are involved in the repair mechanisms of the Topo-DNA complex and would possibly have a further positive effect in apoptosis induction. 42,43 Liposomal formulations could also be possible further approaches for P8-D6. This special drug formulation would significantly increase the selectivity of the active ingredient and thus contribute to better tolerability of the active ingredient, since topoisomerase are ubiquitous in cells and can thus trigger slight side effects there, as known with other cytostatics.

Conclusion
In summary, P8-D6 has promising antitumour properties in 2D, 3D and co-culture in OvCa. It has fewer effects on normal ovarian cells and hepatocytes than its references. To sum up, P8-D6 is a strong and rapid inductor of apoptosis journals.sagepub.com/home/tam 9 and warrants further development. Further in vivo experiments for P8-D6 are needed to verify antitumour effects also for complex multiorgan systems. In addition, further studies on other side effects that could lead to dose-limitation should be performed.