Combined PARP and Dual Topoisomerase Inhibition Potentiates Genome Instability and Cell Death in Ovarian Cancer

Although ovarian cancer is a rare disease, it constitutes the fifth leading cause of cancer death among women. It is of major importance to develop new therapeutic strategies to improve survival. Combining P8-D6, a novel dual topoisomerase inhibitor with exceptional anti-tumoral properties in ovarian cancer and compounds in preclinical research, and olaparib, a PARP inhibitor targeting DNA damage repair, is a promising approach. P8-D6 induces DNA damage that can be repaired by base excision repair or homologous recombination in which PARP plays a major role. This study analyzed benefits of combining P8-D6 and olaparib treatment in 2D and 3D cultures with ovarian cancer cells. Measurement of viability, cytotoxicity and caspase activity were used to assess therapy efficacy and to calculate the combination index (CI). Further DNA damage was quantified using the biomarkers RAD51 and γH2A.X. The combinational treatment led to an increased caspase activity and reduced viability. CI values partially show synergisms in combinations at 100 nM and 500 nM P8-D6. More DNA damage accumulated, and spheroids lost their membrane integrity due to the combinational treatment. While maintaining the same therapy efficacy as single-drug therapy, doses of P8-D6 and olaparib can be reduced in combinational treatments. Synergisms can be seen in some tested combinations. In summary, the combination therapy indicates benefits and acts synergistic at 100 nM and 500 nM P8-D6

ADAM17 Inhibition Increases the Impact of Cisplatin Treatment in Ovarian Cancer Spheroids

Chemotherapy resistance is a major challenge in ovarian cancer (OvCa). Thus, novel treatment combinations are highly warranted. However, many promising drug candidates tested in two-dimensional (2D) cell culture have not proved successful in the clinic. For this reason, we analyzed our drug combination not only in monolayers but also in three-dimensional (3D) tumor spheroids. One potential therapeutic target for OvCa is A disintegrin and metalloprotease 17 (ADAM17). ADAM17 can be activated by chemotherapeutics, which leads to enhanced tumor growth due to concomitant substrate cleavage. Therefore, blocking ADAM17 during chemotherapy may overcome resistance. Here, we tested the effect of the ADAM17 inhibitor GW280264X in combination with cisplatin on ovarian cancer cells in 2D and 3D. In 2D, the effect on five cell lines was analyzed with two readouts. Three of these cell lines formed dense aggregates or spheroids (HEY, SKOV-3, and OVCAR-8) in 3D and the treatment effect was analyzed with a multicontent readout (cytotoxicity, viability, and caspase3/7 activation). We tested the combined therapy on tumor spheroids derived from primary patient cells. In 2D, we found a significant reduction in the half minimal (50%) inhibitory concentration (IC50) value of the combined treatment (GW280264X plus cisplatin) in comparison with cisplatin monotherapy in all five cell lines with both 2D readout assays (viability and caspase activation). In contrast, the combined treatment only showed an IC50 reduction in HEY and OVCAR-8 3D tumor spheroid models using caspase3/7 activity or CelltoxTM Green as the readout. Finally, we found an improved effect of GW280264X with cisplatin in tumor spheroids derived from patient samples. In summary, we demonstrate that ADAM17 inhibition is a promising treatment strategy in ovarian cancer

Investigations of the dual topoisomerase inhibitor P8-D6 for the therapy of gynecological tumors

Die Entwicklung innovativer zytotoxischer Wirkstoffe hat einen hohen klinischen Bedarf in der Krebstherapie. In der gynäkologischen Onkologie ist der Brustkrebs der häufigste und der Eierstockkrebs der aggressivste Tumor. Resistenzentwicklung und schwere Nebenwirkungen sind die Gründe für einen mangelnden Behandlungserfolg. P8-D6 ist ein neuer effektiver Induktor der Apoptose, indem er als dualer Topoisomerase-Inhibitor wirkt. In diesen Untersuchungen wurde die Wirksamkeit von P8-D6 auf Eierstockkrebs- und Brustkrebszellen im 2D-Modell im Vergleich zu Standardtherapeu-tika untersucht. Erweitert durch 3D und Co-Kultur wurden die mit P8-D6 behandelten Zellen auf Veränderungen der Zytotoxizität, der Apoptoserate und der Membranintegri-tät mittels REM untersucht. Ebenso wurden die Auswirkungen von P8-D6 auf die nicht krebsartigen humanen Ovarialoberflächenepithelzellen und primäre humane Hepatozy-ten bestimmt. Die Untersuchungen zeigen bei Eierstockkrebs und Brustkrebs eine signifikante Erhöhung der Apoptose und Zytotoxizität durch P8-D6. Nicht-Krebszellen wurden durch P8-D6 geringfügig beeinflusst. Zusammenfassend lässt sich sagen, dass P8-D6 in vitro ein starker und innovativer Wirkstoff bei Therapie des Eierstockkrebs und Brustkrebs ist.The development of innovative cytotoxic compounds has a high clinical need in cancer therapy. In gynaecological oncology, breast cancer is the most common and ovarian cancer is the most aggressive tumour. Development of resistance and severe side effects are the reasons for a lack in success treatments. P8-D6 is a new effective inductor of apoptosis by acting as a dual topoisomerase inhibitor. In the present study the effective-ness of P8-D6 on ovarian cancer and breast cancer cells were investigated in 2D model compared to standard therapeutic agents. Enhanced by 3D and co-culture, the P8-D6 treated cells were examined for changes in cytotoxicity, apoptosis rate and membrane integrity via SEM. Likewise, the effects of P8-D6 on the non-cancer human ovarian surface epithelial cells and primary human hepatocytes were determined. The studies demonstrate in ovarian cancer and breast cancer a significantly increase of apoptosis and cytotoxicity by P8-D6. Non-cancer cells were slightly affected by P8-D6. In conclu-sion, P8-D6 is in vitro a potent and innovative drug in ovarian cancer and breast cancer therapy

A simple approach for restoration of differentiation and function in cryopreserved human hepatocytes

Primary human hepatocytes are used in all facets of liver research, from in vitro studies of xenobiotic disposition and toxicity to the clinical management of liver failure. Unfortunately, cellular stress during isolation and cryopreservation causes a highly unpredictable loss of the ability to attach and form cell-matrix and cell-cell interactions. Reasoning that this problem could be mitigated at the post-thawing stage, we applied label-free quantitative global proteomics to analyze differences between attached and non-attached fractions of cryopreserved human hepatocyte batches. Hepatocytes that were unable to attach to a collagen matrix showed many signs of cellular stress, including a glycolytic phenotype and activation of the heat shock response, ultimately leading to apoptosis activation. Further analysis of the activated stress pathways revealed an increase in early apoptosis immediately post-thawing, which suggested the possibility of stress reversal. Therefore, we transiently treated the cells with compounds aimed at decreasing cellular stress via different mechanisms. Brief exposure to the pan-caspase apoptosis inhibitor Z-VAD-FMK restored attachment ability and promoted a differentiated morphology, as well as formation of 3D spheroids. Further, Z-VAD-FMK treatment restored metabolic and transport functions, with maintained sensitivity to hepatotoxic insults. Altogether, this study shows that differentiation and function of suboptimal human hepatocytes can be restored after cryopreservation, thus markedly increasing the availability of these precious cells

The Aza-Analogous Benzo[c]phenanthridine P8-D6 Acts as a Dual Topoisomerase I and II Poison, thus Exhibiting Potent Genotoxic Properties

The benzo[c]phenanthridine P8-D6 was recently found to suppress the catalytic activity of both human topoisomerase (Topo) I and II. Concomitantly, potent cytotoxic activity was observed in different human tumor cell lines, raising questions about the underlying mechanisms in vitro. In the present study, we addressed the question of whether P8-D6 acts as a so-called Topo poison, stabilizing the covalent Topo–DNA intermediate, thus inducing fatal DNA strand breaks in proliferating cells. In HT-29 colon carcinoma cells, fluorescence imaging revealed P8-D6 to be taken up by the cells and to accumulate in the perinuclear region. Confocal microscopy demonstrated that the compound is partially located inside the nuclei, thus reaching the potential target. In the “in vivo complex of enzyme” (ICE) bioassay, treatment of HT-29 cells with P8-D6 for 1 h significantly enhanced the proportion of Topo I and II covalently linked to the DNA in concentrations ≥1 µM, indicating effective dual Topo poisoning. Potentially resulting DNA damage was analyzed by single-cell gel electrophoresis (“comet assay”). Already at 1 h of incubation, significant genotoxic effects were observed in the comet assay in concentrations as low as 1 nM. Taken together, the present study demonstrates the high Topo-poisoning and genotoxic potential of P8-D6 in human tumor cells

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

Hepatocyte size fractionation allows dissection of human liver zonation

Human hepatocytes show marked differences in cell size, gene expression, and function throughout the liver lobules, an arrangement termed liver zonation. However, it is not clear if these zonal size differences, and the associated phenotypic differences, are retained in isolated human hepatocytes, the “gold standard” for in vitro studies of human liver function. Here, we therefore explored size differences among isolated human hepatocytes and investigated whether separation by size can be used to study liver zonation in vitro. We used counterflow centrifugal elutriation to separate cells into different size fractions and analyzed them with label-free quantitative proteomics, which revealed an enrichment of 151 and 758 proteins (out of 5163) in small and large hepatocytes, respectively. Further analysis showed that protein abundances in different hepatocyte size fractions recapitulated the in vivo expression patterns of previously described zonal markers and biological processes. We also found that the expression of zone-specific cytochrome P450 enzymes correlated with their metabolic activity in the different fractions. In summary, our results show that differences in hepatocyte size matches zonal expression patterns, and that our size fractionation approach can be used to study zone-specific liver functions in vitro

High Antitumor Activity of the Dual Topoisomerase Inhibitor P8-D6 in Breast Cancer

Breast cancer constitutes the leading cause of cancer deaths among females. However, numerous shortcomings, including low bioavailability, resistance and significant side effects, are responsible for insufficient treatment. The ultimate goal, therefore, is to improve the success rates and, thus, the range available treatment options for breast cancer. Consequently, the identification, development and evaluation of potential novel drugs such as P8-D6 with seminal antitumor capacities have a high clinical need. P8-D6 effectively induces apoptosis by acting as a dual topoisomerase I/II inhibitor. This study provides an overview of the effectiveness of P8-D6 in breast cancer with both 2D monolayers and 3D spheroids compared to standard therapeutic agents. For this drug effectiveness review, cell lines and ex vivo primary cells were used and cytotoxicity, apoptosis rates and membrane integrity were examined. This study provides evidence for a significant P8-D6-induced increase in apoptosis and cytotoxicity in breast cancer cells compared to the efficacy of standard therapeutic drugs. To sum up, P8-D6 is a fast and powerful inductor of apoptosis and might become a new and suitable therapeutic option for breast cancer in the future

Nectin-4 as Blood-Based Biomarker Enables Detection of Early Ovarian Cancer Stages

Ovarian cancer is the third most common gynecological malignancy and has the highest mortality rate. Owing to unspecific symptoms, ovarian cancer is not detected until an advanced stage in about two-thirds of cases. Therefore, it is crucial to establish reliable biomarkers for the early stages to improve the patients’ prognosis. The aim of this study is to investigate whether the ADAM17 substrates Nectin-4, Heparin-binding EGF-like growth factor (HB-EGF) and Amphiregulin (AREG) could function as potential tumor markers for ovarian cancer. In this study a set of 231 sera consisting of 131 ovarian cancer patients and 100 healthy age-matched controls were assembled. Nectin-4, HB-EGF and AREG levels of preoperatively collected sera were determined by enzyme-linked immunosorbent assay (ELISA). Our analysis revealed that Nectin-4 and HB-EGF were significantly increased compared to the age-matched control group (p < 0.0001, p = 0.016). Strikingly, significantly higher Nectin-4 and HB-EGF levels were detected in early-stage FIGO I/II (p <0.001; p = 0.025) compared to healthy controls. Eighty-four percent (16/19) of patients with low Ca-125 levels showed increased Nectin-4 levels. Our study proposes Nectin-4 and HB-EGF as promising blood-based biomarkers for the detection of early stages of ovarian cancer patients that would not have been detected by Ca-125

ADAM17—A Potential Blood-Based Biomarker for Detection of Early-Stage Ovarian Cancer

Ovarian cancer has the highest mortality rate among gynecological tumors. This is based on late diagnosis and the lack of early symptoms. To improve early detection, it is essential to find reliable biomarkers. The metalloprotease ADAM17 could be a potential marker, as it is highly expressed in many solid tumors, including ovarian and breast cancer. The aim of this work is to evaluate the relevance of ADAM17 as a potential diagnostic blood-based biomarker in ovarian cancer. Ovarian cancer cell lines IGROV-1 and A2780, as well as primary patient-derived tumor cells obtained from tumor tissue and ascitic fluid, were cultured to analyze ADAM17 abundance in the culture supernatant. In a translational approach, a cohort of 117 well-characterized ovarian cancer patients was assembled and ADAM17 levels in serum and corresponding ascitic fluid were determined at primary diagnosis. ADAM17 was quantified by enzyme-linked immunosorbent assay (ELISA). In the present study, ADAM17 was detected in the culture supernatant of ovarian cancer cell lines and primary cells. In addition, ADAM17 was found in serum and ascites of ovarian cancer patients. ADAM17 level was significantly increased in ovarian cancer patients compared to an age-matched control group (p < 0.0001). Importantly early FIGO I/II stages, which would not have been detected by CA-125, were associated with higher ADAM17 concentrations (p = 0.007). This is the first study proposing ADAM17 as a serum tumor marker in the setting of a gynecological tumor disease. Usage of ADAM17 in combination with CA-125 and other markers could help detect early stages of ovarian cancer