Filiz Senbabaoglu

The magnitude of gonadotoxicity of chemotherapy drugs on ovarian follicles and granulosa cells varies depending upon the category of the drugs and the type of granulosa cells

STUDY QUESTION Do different chemotherapy drugs exert the same magnitude of cytotoxicity on dormant primordial follicles and the growing follicle fraction in the ovary in vivo and on mitotic non-luteinized and non-mitotic luteinized granulosa cells in vitro? SUMMARY ANSWER Cyclophosphamide (alkylating agent) and cisplatin (alkylating like) impacted both primordial and pre-antral/antral follicles and both mitotic and non-mitotic granulosa cells, whereas the anti-metabolite cancer drug gemcitabine was detrimental only to pre-antral/antral follicles and mitotic non-luteinized granulosa cells. WHAT IS KNOWN ALREADY It is already known that anti-metabolite cancer drugs are less detrimental to the ovary than alkylating and alkylating like agents, such as cyclophosphamide and cisplatin. This assumption is largely based on the results of clinical reports showing lower rates of amenorrhea in women receiving anti-metabolite agent-based regimens compared with those treated with the protocols containing an alkylating drug or a platinum compound. But a quantitative comparison of gonadotoxicity with a histomorphometric proof of evidence has not been available for many chemotherapy drugs. Therefore, we combined in this study in vivo and in vitro models of human and rat origin that allows a comparative analysis of the impact of different chemotherapy agents on the ovary and granulosa cells using real-time quantitative cell indices, histomorphometry, steroidogenesis assays, and DNA damage and cell death/viability markers. We also aimed to investigate if there is a difference between mitotic and non-mitotic granulosa cells in terms of their sensitivity to the cytotoxic actions of chemotherapy drugs with different mechanisms of action. This issue has not been addressed previously. STUDY DESIGN, SIZE, DURATION This translational research study involved in vivo analyses of ovaries in rats and in vitro analyses of granulosa cells of human and rat origin. PARTICIPANTS/MATERIALS, SETTING, METHODS For the in vivo assays, 54 4- to 6-week old Sprague-Dawley young female rats were randomly allocated into four groups of 13 to receive a single IP injection of: saline (control), gemcitabine (200 mg/kg), cisplatin (50 mg/kg) or cyclophosphamide (200 mg/kg). The animals were euthanized 72 h later. Follicle counts and serum AMH levels were compared between the groups. In vitro cytotoxicity studies were performed using mitotic non-luteinized rat (SIGC) and human (COV434, HGrC1) granulosa cells, and non-mitotic luteinized human (HLGC) granulosa cells. The cells were plated at a density of 5000 cells/well using DMEM-F12 culture media supplemented with 10% FBS. Chemotherapy agents were used at their therapeutic blood concentrations. The growth of mitotic granulosa cells was monitored real-time using xCelligence system. Live/dead cell and apoptosis assays were also carried out using intravital Yo-Pro-1 staining and cleaved caspase-3 expression, respectively. Estradiol (E2), progesterone (P) and anti-Mullerian hormone (AMH) levels were assayed with ELISA. MAIN RESULTS AND THE ROLE OF CHANCE Cyclophosphamide and cisplatin caused massive atresia of both primordials and growing follicles in the rat ovary whereas gemcitabine impacted pre-antral/antral follicles only. Cyclophosphamide and cisplatin induced apoptosis of both mitotic non-luteinized and non-mitotic luteinized granulosa cells in vitro. By contrast, cytotoxicity of gemcitabine was confined to mitotic non-luteinized granulosa cells. LIMITATIONS, REASONS FOR CAUTION This study tested only three chemotherapeutic agents. The experimental methodology described here could be applied to other drugs for detailed analysis of their ovarian cytotoxicity. WIDER IMPLICATIONS OF THE FINDINGS These findings indicate that in vivo and in vitro cytotoxic actions of chemotherapy drugs on the ovarian follicles and granulosa cells vary depending upon the their mechanism of action and the nature of the granulosa cells.

GnRH agonist leuprolide acetate does not confer any protection against ovarian damage induced by chemotherapy and radiation in vitro

STUDY QUESTION Is there any in vitro evidence for or against ovarian protection by co-administration of a GnRH agonist with chemotherapy in human? SUMMARY ANSWER The co-administration of GnRH agonist leuprolide acetate with cytotoxic chemotherapy agents does not preserve ovarian reserve in vitro. WHAT IS KNOWN ALREADY Randomized controlled trials of the co-administration of gonadotrophin-releasing hormone (GnRH) agonists with adjuvant chemotherapy to preserve ovarian function have shown contradictory results. This fact, together with the lack of a proven molecular mechanism of action for ovarian protection with GnRH agonist (GnRHa) places this approach as a fertility preservation strategy under scrutiny. We therefore aimed in this study to provide in vitro evidence for or against the role of GnRHa in the prevention of chemotherapy-induced damage in human ovary. STUDY DESIGN, SETTINGS, SIZE AND DURATION This translational research study of ex vivo and in vitro models of human ovary and granulosa cells was conducted in a university hospital between 2013 and 2015. PARTICIPANTS/MATERIALS, SETTING, METHODS Ovarian cortical pieces (n = 15, age 14-37) and mitotic non-luteinized (COV434 and HGrC1) and non-mitotic luteinized human granulosa cells (HLGC) expressing GnRH receptor were used for the experiments. The samples were treated with cyclophosphamide, cisplatin, paclitaxel, 5-FU, or TAC combination regimen (docetaxel, adriamycin and cyclophosphamide) with and without GnRHa leuprolide acetate for 24 h. DNA damage, apoptosis, follicle reserve, hormone markers of ovarian function and reserve (estradiol (E2), progesterone (P) and anti-mullerian hormone (AMH)) and the expression of anti-apoptotic genes (bcl-2, bcl-xL, bcl-2L2, Mcl-1, BIRC-2 and XIAP) were compared among control, chemotherapy and chemotherapy + GnRHa groups. MAIN RESULTS AND THE ROLE OF CHANCE The greatest magnitude of cytotoxicity was observed in the samples treated with cyclophosphamide, cisplatin and TAC regimen. Exposure to these drugs resulted in DNA damage, apoptosis and massive follicle loss along with a concurrent decline in the steroidogenic activity of the samples. GnRHa co-administered with chemotherapy agents stimulated its receptors and raised intracellular cAMP levels. But it neither activated anti-apoptotic pathways nor prevented follicle loss, DNA damage and apoptosis induced by these drugs. LIMITATIONS, REASONS FOR CAUTION Our findings do not conclusively rule out the possibility that GnRHa may offer protection, if any, through some other mechanisms in vivo. WIDER IMPLICATIONS OF THE FINDINGS GnRH agonist treatment with chemotherapy does not prevent or ameliorate ovarian damage and follicle loss in vitro. These data can be useful when consulting a young patient who may wish to receive GnRH treatment with chemotherapy to protect her ovaries from chemotherapy-induced damage.

Identification of Mitoxantrone as a TRAIL-sensitizing agent for Glioblastoma Multiforme

ABSTRACT Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) has tremendous promise in treating various forms of cancers. However, many cancer cells exhibit or develop resistance to TRAIL. Interestingly, many studies have identified several secondary agents that can overcome TRAIL resistance. To expand on these studies, we conducted an extensive drug-re-profiling screen to identify FDA-approved compounds that can be used clinically as TRAIL-sensitizing agents in a very malignant type of brain cancer, Glioblastoma Multiforme (GBM). Using selected isogenic GBM cell pairs with differential levels of TRAIL sensitivity, we revealed 26 TRAIL-sensitizing compounds, 13 of which were effective as single agents. Cardiac glycosides constituted a large group of TRAIL-sensitizing compounds, and they were also effective on GBM cells as single agents. We then explored a second class of TRAIL-sensitizing drugs, which were enhancers of TRAIL response without any effect on their own. One such drug, Mitoxantrone, a DNA-damaging agent, did not cause toxicity to non-malignant cells at the doses that synergized with TRAIL on tumor cells. We investigated the downstream changes in apoptosis pathway components upon Mitoxantrone treatment, and observed that Death Receptors (DR4 and DR5) expression was upregulated, and pro-apoptotic and anti-apoptotic gene expression patterns were altered in favor of apoptosis. Together, our results suggest that combination of Mitoxantrone and TRAIL can be a promising therapeutic approach for GBM patients.

Cytotoxicity and mitogenicity assays with real-time and label-free monitoring of human granulosa cells with an impedance-based signal processing technology intergrating micro-electronics and cell biology.

A recently developed technology (xCelligence) integrating micro-electronics and cell biology allows real-time, uninterrupted and quantitative analysis of cell proliferation, viability and cytotoxicity by measuring the electrical impedance of the cell population in the wells without using any labeling agent. In this study we investigated if this system is a suitable model to analyze the effects of mitogenic (FSH) and cytotoxic (chemotherapy) agents with different toxicity profiles on human granulosa cells in comparison to conventional methods of assessing cell viability, DNA damage, apoptosis and steroidogenesis. The system generated the real-time growth curves of the cells, and determined their doubling times, mean cell indices and generated dose-response curves after exposure to cytotoxic and mitogenic stimuli. It accurately predicted the gonadotoxicity of the drugs and distinguished less toxic agents (5-FU and paclitaxel) from more toxic ones (cisplatin and cyclophosphamide). This platform can be a useful tool for specific end-point assays in reproductive toxicology.

KDM2B, an H3K36-specific demethylase, regulates apoptotic response of GBM cells to TRAIL

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can selectively kill tumor cells. TRAIL resistance in cancers is associated with aberrant expression of the key components of the apoptotic program. However, how these components are regulated at the epigenetic level is not understood. In this study, we investigated novel epigenetic mechanisms regulating TRAIL response in glioblastoma multiforme (GBM) cells by a short-hairpin RNA loss-of-function screen. We interrogated 48 genes in DNA and histone modification pathways and identified KDM2B, an H3K36-specific demethylase, as a novel regulator of TRAIL response. Accordingly, silencing of KDM2B significantly enhanced TRAIL sensitivity, the activation of caspase-8, -3 and -7 and PARP cleavage. KDM2B knockdown also accelerated the apoptosis, as revealed by live-cell imaging experiments. To decipher the downstream molecular pathways regulated by KDM2B, levels of apoptosis-related genes were examined by RNA-sequencing upon KDM2B loss, which revealed derepression of proapoptotic genes Harakiri (HRK), caspase-7 and death receptor 4 (DR4) and repression of antiapoptotic genes. The apoptosis phenotype was partly dependent on HRK upregulation, as HRK knockdown significantly abrogated the sensitization. KDM2B-silenced tumors exhibited slower growth in vivo. Taken together, our findings suggest a novel mechanism, where the key apoptosis components are under epigenetic control of KDM2B in GBM cells.

Endogenous c-Jun N-terminal kinase (JNK) activity marks the boundary between normal and malignant granulosa cells

Granulosa cell tumor of the ovary (GCT) is a very rare tumor, accounting for only 2% of all ovarian tumors. It originates from sex cords in the ovary and can be divided into adult (95%) and juvenile (5%) types based on histologic findings. To date, no clear etiologic process has been identified other than a missense point mutation in the FOXL2 gene. Our previous works showed that c-Jun N-terminal kinase (JNK) pathway plays critical role in cell cycle progression and mitosis of normal and immortalized granulosa cells and follicle growth in rodent ovaries. These findings led us to investigate the role of JNK pathway in the granulosa cell tumor of the ovary. We used two different GCT cell lines (COV434 and KGN) and fresh GCT samples of adult and juvenile types obtained from the patients during surgery. We have discovered that endogenous kinase activity of JNK is markedly enhanced in the GCT samples and cell lines, whereas it was almost undetectable in mitotic non-malignant human granulosa cells. The inhibition of JNK pathway in GCT cell lines with two different pharmacologic inhibitors (SP600125 and AS601245) or siRNA resulted in a dose-dependent reduction in in vitro cell growth, increased apoptosis and diminished estradiol and AMH productions. JNK inhibition was also associated with a decrease in the number of cells positive for mitosis marker phospho-histone H3Ser 10 in the asynchronous cells; and diminished EdU uptake during S phase and cell cycle arrest at G2/M-phase transition in the synchronized cells. Ex vivo treatment of patient-derived GCT samples with JNK inhibitors for 24 h significantly decreased their in vitro growth and estradiol and AMH productions. Furthermore, in human GCT xenograft model, in vivo tumor growth was significantly reduced and plasma AMH levels were significantly decreased in SCID mice after administration of JNK inhibitors and siRNA. These findings suggest that targeting JNK pathway may provide therapeutic benefit in the treatment of granulosa cell tumors for which currently no curative therapy exists beyond surgery.

A platinum blue complex exerts its cytotoxic activity via DNA damage and induces apoptosis in cancer cells

Here, we describe the characteristics of a Pt‐blue complex [Pt4(2‐atp)8(H2O)(OH)] (2‐atp: 2‐aminothiophenol) as a prodrug for its DNA‐binding properties and its use in cancer therapy. The nature of the interaction between the Pt‐blue complex and DNA was evaluated based on spectroscopic measurements, the electronic absorption spectra, thermal behavior, viscosity, fluorometric titration, and agarose gel electrophoresis. Our results suggested that the compound was able to partially intercalate DNA and appeared to induce both single‐ and double‐stranded breaks (DBS) on DNA in vitro, but no DSBs in cells. The ability of the compound to induce DNA damage was dependent on reactive oxygen species (ROS) in vitro. There was also elevated formation of ROS and SOD expression in response to drug treatment in cell culture. The complex was found to be more cytotoxic to cancer cells in comparison with noncancer controls using WST‐1 assay. The mean of cell death was determined to be apoptosis as assessed via biochemical, morphological, and molecular observations, including DNA condensation/fragmentation analysis, live cell imaging microscopy, TUNEL analyses, and increase in the levels of pro‐apoptotic genes such as Bag3, Bak, Bik, Bmf, and Hrk. Hence, the Pt‐blue complex under study grants premise for further studies.

PO-306 Identification of chromatin modifiers regulating temozolomide resistance in glioblastoma multiforme

Introduction Glioblastoma multiforme (GBM) is the most common of all malignant brain tumours. Unfortunately, only 5.1% of patients survive five years post diagnosis. Therapy options for GBM patients are very limited, majority of the patients receive radiation therapy and the chemotherapeutic Temozolomide (TMZ). However, most tumours recur making therapy resistance an extremely important issue. Cancers show aberrant global epigenetic alterations, yet, the epigenetic mechanisms that cause therapy resistance are not well-known. In this project, the role of chromatin modifying proteins in TMZ-resistance is studied by a chemical screen. Material and methods We established TMZ resistant cell lines by two different methodologies, 1) dose escalation method and 2) high dose TMZ selection. For the dose escalation method, U373 cells were treated TMZ every two days, starting from 25 µM. Every two weeks, dosage was doubled (or when cells become resistant to that dosage) up to 250 µM. For high dose TMZ resistant selection, cells were treated with 250 µM TMZ for 15 days. Both cells lines have been kept under TMZ treatment for 3 months. To identify chemical inhibitors that target resistant cells, the TMZ-resistant cells and their parental controls were treated with a chemical library consisting of 90 epigenetic drugs against chromatin modifiers and cell viability was measured after 72 hours. Results and discussions We successfully generated isogenic subpopulations of GBM cell lines that are resistant to TMZ. We observed that the high dosage TMZ treatment regimen caused a more sustainable resistant cell line compared to dose escalation regimen. Both resistant cell line models were stable, as they did not depend on prolonged TMZ treatment and remained resistant to TMZ after 3 months of drug holiday. We then screened for epigenetic compounds that target TMZ-resistant cells. In addition to PARP inhibitors, Olaparib and Rucaparib, we identified Histone Deacetylase (HDAC) inhibitors as TMZ-sensitising agents. We are currently delineating the epigenetic alterations between parental and TMZ-resistant cells and assess the role of HDAC-mediated changes in TMZ-response by RNA-seq. Conclusion Histone acetylation and deacetylation are interesting focus areas in drug resistance because of the central role of these modifications in many aspects of cell physiology and pathology. In our screen, we have shown that various HDAC inhibitors can sensitise GBM cells to TMZ in established cells as well as our newly generated in acquired TMZ resistance models.

Abstract B73: Screen among 1200 FDA-approved drug library reveals mitoxantrone as a TRAIL-sensitizing agent for glioblastoma multiforme

Glioblastoma multiforme (GBM) is the most aggressive and frequent type of primary brain tumor with dismal survival rates. As GBM cells suppress apoptosis and evade death, re-activating dormant apoptotic programs with pro-apoptotic ligands or small molecules might be a promising approach to direct tumor cells to self-destruct. As such, the tumor-selective killing capacity of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) makes it a potential treatment option in GBM. However, many tumor cells are intrinsically resistant and/or acquire resistance to TRAIL. While the mechanisms of TRAIL resistance is still not completely solved, several studies suggest that TRAIL resistance can be overcome by combination therapies. Therefore, combination of TRAIL with secondary chemotherapeutics, radiation or other novel therapeutic drugs might be a favorable therapeutic strategy. In this study, we aimed to identify TRAIL sensitizing agents and utilized a chemical library consisting of 1200 Food and Drug Administration (FDA)-approved compounds in a high-throughput screen that assessed GBM cell viability. In the screen that was conducted in TRAIL-midsensitive and TRAIL-resistant isogenic cell pairs in parallel, we revealed 26 TRAIL-sensitizing compounds, 13 of which were effective in reducing cell viability as single agents. Cardiac glycosides constituted a large group among our hits. While cardiac glycosides were potent TRAIL-sensitizers and also singly effective on GBM cells, they caused cell death in non-malignant cells, such as GBM-patient derived fibroblasts. Therefore, the future use of these agents might be challenging due to toxicity problems. We then explored the second class of TRAIL-sensitizing drugs, which were enhancers of TRAIL response without any effect on their own. One such drug, Mitoxantrone, a DNA damaging agent, did not cause any toxicity to non-malignant cells at the doses that were able to synergize with TRAIL on tumor cells. We then investigated the downstream changes in apoptosis pathway components upon Mitoxantrone treatment, and observed that Death Receptor (DR4 and DR5) expression was upregulated markedly, as revealed by qRT-PCR and Western blotting. In addition, we observed significant changes in pro-apoptotic and anti-apoptotic gene expression. Together, our results suggest that combination of Mitoxantrone and TRAIL can be a promising therapeutic approach for GBM patients. Citation Format: Filiz Senbabaoglu, Ahmet Cingoz, Ezgi Kaya, Selena Kazancioglu, Nathan Alan Lack, Ceyda Acilan, Tugba Bagci-Onder. Screen among 1200 FDA-approved drug library reveals mitoxantrone as a TRAIL-sensitizing agent for glioblastoma multiforme. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B73.