Arch Gynecol Obstet (2015) 291:131–141 DOI 10.1007/s00404-014-3389-z
GYNECOLOGIC ONCOLOGY
Anti-tumour activity of phosphoinositide-3-kinase antagonist AEZS-126 in models of ovarian cancer Jens C. Hahne • Antje Kurz • Susanne R. Meyer Johannes Dietl • Jo¨rg B. Engel • Arnd Honig
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Received: 3 March 2014 / Accepted: 21 July 2014 / Published online: 13 August 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract Purpose Platinum resistance is the most crucial problem for treatment of ovarian cancer. There is a clinical need for new treatment strategies which overcome platinum resistance. Recently high level of AKT was shown to be involved in platinum resistance and furthermore in resistance against Natural-killer (NK)-cell mediated killing in ovarian cancer. Methods Here, we investigate the ability of the PI3K/ AKT inhibitor AEZS-126 alone and in combination with rapamycin to selectively target ovarian cancer cell proliferation and survival in vitro by MTT-assays and FACS based analysis. Furthermore the mechanism of cytotoxicity is analysed by FACS based assays. The NK-killing efficiency of ovarian cancer cells with and without pre-treatment with AEZS-126 was analysed. Results AEZS-126 showed good anti-tumour activity in in vitro models of ovarian cancer. Main mechanism of cytotoxicity seems to be necroptosis which could be abrogated by co-incubation with necrostatin-1. Furthermore pre-treatment of platinum resistant cells with AEZS126 resulted in an increased accessibility of these tumour cells for killing by NK-cells.
J. C. Hahne and A. Kurz have contributed equally to this work. J. C. Hahne A. Kurz S. R. Meyer J. Dietl A. Honig (&) Department of Gynecology, University Hospital of Wu¨rzburg, Josef-Schneider-Str. 4, 97080 Wu¨rzburg, Germany e-mail: [email protected] J. C. Hahne e-mail: [email protected] J. B. Engel Department of Gynecology, University of Regensburg, Landshuter Str. 65, 93055 Regensburg, Germany
Conclusion We demonstrated the highly efficient antitumour activity of AEZS-126 in in vitro models of ovarian cancer. Due to the good anti-tumour activity and the expected increase in NK-cell mediated killing even of platinum resistant tumour cells, AEZS-126 seems to be a promising candidate for clinical testing in ovarian cancer. Keywords Ovarian cancer AKT Platinum-resistance NK-cells
Introduction Ovarian cancer is the most common cause of cancer death from gynecologic tumours and currently causes about 100,000 deaths per year world wide [1]. In 2009 a total of 7,430 women were diagnosed with ovarian cancer in Germany [2]. The relative 5-year survival for ovarian cancer is only 40 % in Germany [2]. While treatment of ovarian cancer with platinum-based agents has been established for decades, these still remain the most active substances for this entity [3]. Accordingly, primary resistance to platinum-based therapy is associated with a worse disease free and overall survival [4]. However, virtually all patients eventually develop secondary resistance to platinum based agents and compounds used for second or third line treatment. Other substances display substantially less anti-cancer activity as compared to platinum [5]. There is a definite clinical need to develop new treatment strategies to overcome platinum resistance. As survival is strongly influenced by immunological parameters, immunotherapeutic strategies appear promising; therefore a better understanding of the interaction between ovarian tumour cells and cells of the immune system is necessary.
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Natural-killer (NK)-cells play an important role in immune surveillance and coordinating responses of other immune cells. Most tumour cells express surface molecules that can be recognised by activating receptors on NK-cells [6]. The expression of these receptors make such cells susceptible to endogenous NK-cells, but malignant cells have developed mechanisms to evade innate immune surveillance [7–9]. In patients with cancer, it is presumed that tumour cells have developed mechanisms to suppress NKcell activation and resist lysis by endogenous NK-cells, but the molecular basis for tumour cell resistance against this lysis is not well understood. Alterations of the serine/threonine kinase AKT/PKB (protein kinase B) pathway have been detected in several human malignancies including ovarian cancer [9]. AKT has a broad range of downstream effectors that regulate cell processes such as cell growth, cell cycle progression, survival, migration, and angiogenesis [11]. The AKT pathway is a promising target for cancer therapy, as it is a main nodal point where extracellular and intracellular oncogenic signals are integrated. Due to the key role of AKT in malignant transformation numerous inhibitors of the AKTpathway have been developed, and are currently in various stages of clinical development [12]. In human specimens of ovarian cancer AKT was found to be activated in 68 % [10] and PI3K (phosphoinositide-3kinase), an upstream component of the AKT-pathway, was found to be mutated in 12 % of the cases [13]. Recent evidence by our group and others has shown that overactivation of the AKT-pathway may be associated with platinum resistance [14–19]. It was demonstrated that parental A2780 cells become platinum resistant by overexpression of AKT and that platinum resistance in Acis2780 cells can be overcome by transfection with siRNA downregulating AKT [18]. Furthermore recently it was shown that ovarian tumour cells with an elevated AKT expression levels are protected against NK-cell mediated killing [19]. Because of the well-known fact that combination therapy has the potential to overcome drug resistance or escape from oncogene addiction [16, 20] we evaluated in this work here not only the ability of the PI3K/AKT inhibitor AEZS-126 alone but also in combination with rapamycin to selectively target ovarian cancer cell proliferation and survival in vitro. The connections between the PI3K and mTOR (mammalian target of rapamycin) kinases are multiple and complex, including common substrates, negative feedback loops, or direct activation mechanisms [21]. The mammalian target of rapamycin (mTOR) and the PI3K signaling pathways are commonly deregulated in cancers and promote cellular growth, proliferation, and survival [21]. Second the possibility to recover the NK-cell recognition of cis-platinum resistant ovarian cancer cells by
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inhibition of the PI3K/AKT pathway in these tumour cells was analysed.
Materials and methods Reagents and cell lines AEZS-126 was kindly provided by Aeterna Zentaris GmbH (Frankfurt, Germany) and rapamycin was purchased from Sigma-Aldrich (St. Louis, MO, USA). A2780 and Acis2780 cell lines (both are p53 and KRAS wild-type cell lines) were obtained from ECACC (European Collection of Cell Cultures; Salisbury, UK). The cis-platinum-resistant Acis2780 cell line has been developed by chronic exposure of the parental cis-platinum-sensitive A2780 cell line to increasing concentrations of cis-platinum [22]. Cytotoxicity MTT assay To quantify the cytotoxicity of AEZS-126 the viability of cells was measured with a non-radioactive cell counting assay. Therefore cells were cultured in 96-well flat-bottom plates, in humidified 37 °C and 5 % CO2 atmosphere. The cell density was initially adjusted to 2 9 105 cells/ml in a final volume of 50 ll/well. Cells were treated with different concentrations of AEZS 126 as indicated for 24, 48 and 72 h respectively. For the last 4 h of incubation, cells were pulsed with 10 ll of tetrazolium salt [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT] labelling reagent (Roth, Karlsruhe, Germany) at a final concentration of 0.5 mg/ml. The colorimetric assay is based on the cleavage of yellow MTT to pure violet formazan crystals by metabolic active cells [23]. The crystals were solubilised by addition of 100 ll 10 % SDS in 0.01 M HCl to each well. Absorbance was measured spectrophotometrically using a 540-nm wavelength ELISA reader (Tecan, Ma¨nnedorf, Switzerland) and Magellan software. The experiments were performed in triplicates, and at least three independent experiments were performed for each cell line. Preparation of cell lysates and Western blotting Preparation of cell lysates was performed as previously described [18]. Membranes were probed overnight with anti-Phospho-Akt-antibody and anti-Akt-antibody from Epitomics (Burlingame, CA, USA) and anti-b-actin-antibody from Abcam (Cambridge, UK), respectively. Secondary horseradish peroxidase (HRP)-conjugated antibodies were from Cell Signaling (Frankfurt, Germany). The chemiluminescent HRP substrate solution (Millipore, Schwalbach, Germany) was used for detection.
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NK-cell preparation and lysis assay
in binding buffer at a cell density of 1 9 106 cells/ml. 5 ll of FITC-conjugated Annexin-V were added to 100 ll of the cell suspension and incubated 15 min at room temperature. Than the cells were washed with binding buffer and resuspended in 200 ll binding buffer. After addition of 5 ll propidium iodide (2 lg/ml) the cells were analysed by flow cytometry on a FACSCalibur (Becton–Dickinson, Heidelberg, Germany).
PBMC (primary blood mononuclear cells) were obtained from healthy volunteers by density gradient centrifugation (Biocoll; Biochrom AG, Berlin, Germany). Monocytes were depleted by adherence and the remaining nonadherent PBL were further cultured on irradiated (30 Gy) RPMI8866 feeder cells to obtain polyclonal NK-cell populations [24]. After 6 days of co-culture 500 U IL-2 (Interleukin-2; Peprotech, Hamburg, Germany) were added per ml and 48 h later the polyclonal NK-cell population (effector cells) was used in different killing assays. Therefore the NK-cells were labeled with eFluor 670 (eBioscience, Frankfurt, Germany) and lytic activity against CFSE (Carboxyfluoresceinsuccininidylester)stained (eBioscience, Frankfurt, Germany) tumour cells (targets; 105 cells/well) was assessed in a modified 5 h FATAL assay using various effector:target ratios [25]. Cells were detached by trypsinisation and target cell lysis was determined by flow cytometric analysis of 30,000 target cells in a FACScan flow cytometer (Calibur, BD Biosciences, Heidelberg, Germany). eFluor 670-negative target cells were selected by gating and the percentage of CFSE cells within this population was determined. Spontaneous leakage of CFSE was determined by incubating the target cells with medium alone. Flow cytometry For cell cycle analysis, cells were treated with AEZS-126 as indicated, harvested, fixed and permeabilized over night in ice-cold 70 % ethanol (Merck, Darmstadt, Germany). The cells were washed twice with PBS (phosphate buffered saline). RNA was digested with RNase A (Gibco Life Technologies, Paisley, UK). The DNA was stained with propidium iodide (50 lg/ml). Fluorescence was recorded in a FACSCalibur (Becton–Dickinson, Heidelberg, Germany). Instrument settings were adjusted to move the G0/ G1 peak to 200 relative fluorescence units. Cells to the left of this peak appeared to have DNA content below 2n, indicative of cell death. Aggregated cells were gated out. A total of 2 9 104 cells per condition were recorded.
Apoptosis assay Cellular apoptosis was measured by Annexin-V and propidium iodide staining using Annexin-V Apoptosis Detection Kit FITC (eBioscience, Frankfurt, Germany) according to the manufacturer’s protocol. Briefly, cells were treated with AEZS-126 as indicated, harvested, washed once with binding buffer (10 mM HEPES/NaOH, (pH 7.4), 140 mM NaCl, 2.5 mM CaCl2) and resuspended
Results First of all the capacity of AEZS-126 to inhibit the growth of human ovarian cancer cells was evaluated. Therefore A2780 and Acis2780 cells were chosen, because these cell lines have been used extensively in preclinical studies of ovarian cancer and have been shown to translate well to clinical results. Furthermore the cis-platinum resistant Acis2780 cells and the parental A2780 cells have the same genetic background but differ in regard to platinum resistance. The two human ovarian cancer cell lines were treated with increasing concentrations of PI3K inhibitor AEZS-126 or with solvent only for 24, 48 and 72 h. All experiments were repeated at least in triplicates. AEZS-126 displayed strong growth-inhibitory effects in both ovarian cancer cell lines that became more pronounced with increasing treatment time (Fig. 1a, b). The half maximal inhibitory concentration (IC50) values were approximately 3.5 lM for A2780 and 5 lM for Acis2780 after 24-hincubation. IC50 values at 48 and 72 h were approximately 2.5 lM for A2780 and 1 lM for Acis2780 (Fig. 1a, b). To analyse if AEZS-126 displays antiproliferative effects in metabolically inactive cells, we treated both cell lines with the compound while cultivating them in serumfree medium, i.e. medium devoid of growth factors. Increasing concentrations of AEZS-126 which ranged between 0.2 and 200 lM were used; however, no reduction of the cytotoxicity of the drug was observed (data not shown). Next the efficacy of rapamycin a well established mTOR inhibitor was evaluated. Both ovarian cancer cell lines were treated with increasing concentrations of rapamycin or with solvent only for 24, 48 and 72 h. All experiments were repeated at least in triplicates. As expected rapamycin displayed strong growth-inhibitory effects in both ovarian cancer cell lines that became more pronounced with increasing treatment time (Fig. 1c, d). IC50 values were approximately 13 lM for A2780 and 18 lM for Acis2780 after 24-h-incubation (Fig. 1c, d). IC50 values at 48 h were approximately 2.5 lM for A2780 and 0.5 lM for Acis2780. If cells were incubated for 72 h we found IC50 of approximately 0.08 lM for A2780 and 0.1 lM for Acis2780 (Fig. 1c, d).
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As observed previously for AEZS-126 increasing concentrations of rapamycin which ranged between 0.1 and 20 lM also showed no antiproliferative effects in metabolically inactive cells (data not shown). Finally the parental A2780 and the cis-platinum resistant Acis2780 cells were treated with AEZS-126 and rapamycin in different combinations or with solvents only for 24, 48 and 72 h. All experiments were repeated at least in triplicates. The combination displayed strong growth-inhibitory effects in both ovarian cancer cell lines (Fig. 2). Adding rapamycin to AEZS-126 clearly increased its cytotoxic effect in all cell lines over the whole dose range. Adding rapamycin to AEZS-126 strongly increased its cytotoxic effect in A2780 cells (Fig. 2a, c, e), resulting in a growth inhibition by 20–40 % with rapamycin. In contrast in Acis2780 cells, addition of rapamycin to AEZS-126 resulted in a decreased viability of up to 60–90 %. These very strong effects were observed in the cis-platinum resistant cells by co-treatment with 0.2 lM AEZS-126 and 20 lM rapamycin at all time points (Fig. 2b, d, f). To assess whether AEZS-126 (Fig. 3a, b) and rapamycin (Fig. 3c, d) as single agent or in combination (Fig. 3e, f) arrested cell growth in a specific phase of the cell cycle or rather caused cell death in the ovarian cancer cell lines, we performed a flow cytometric DNA cell cycle analysis on inhibitor-treated A2780 (Fig. 3a, c, e) and Acis2780 cells
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(Fig. 3b, d, f). This revealed in A2780 cells for AEZS-126 a dose-dependent decrease of cells in the G0/G1 and G2/M phases of the cell cycle, whereas the fraction of hypodiploid cells appearing to the left of the G0 cell population was concomitantly increased (Fig. 3a). Since this subG0 population is indicative of cell death, it can be concluded that AEZS-126 not only arrests the growth of A2780 cells, but also actually kills A2780 tumour cells irrespective of their current state in the cell cycle. In contrast AEZS-126 induced in Acis2780 cells only a slight decrease in the G0/ G1 and G2/M phases of the cell cycle as well as a slight increase in subG0 population and S phase (Fig. 3b). This observation argues for cell growth arrest in G0/G1 and G2/ M phase of cell cycle. Rapamycin induced in both cell lines a strong increase in subG0 population and a decrease of cells in the G0/G1 and G2/M phases of the cell cycle (Fig. 3c, d). Co-incubation of the AEZS-126 concentrations representing IC50 for each cell line with increasing amounts of rapamycin resulted in both cell lines in an increased subG0 population (Fig. 3e, f). Whereas for the parental cell line A2780 a decrease of cells in the G0/G 1 and G2/M phases of the cell cycle can be observed, we found in the cis-platinum resistant cell line Acis2780 a decrease of cells in the G2/M phase of the cell cycle and an nearly unaffected amount of cells in G0/G1 phase. So it can be concluded that the combination of AEZS-126 and rapamycin actually kills A2780 cells irrespective of their
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current state in the cell cycle and in the case of Acis2780 cells the cells are arrested in the G0/G1 phase. Next we focused on the mode of action of AEZS-126 in both ovarian cancer cell lines. To enlighten the molecular mechanism of growth inhibition the presence of phosphorylated AKT (pAKT) and unphosphorylated AKT (AKT) were assessed by western blotting in both cell lines that had been treated with the indicated amounts of AEZS126 for 24 h (Fig. 4). AEZS-126 was added to every cell culture so that the concentrations represented 0.5 IC50 and IC50 for each cell line. Specific antibody reactivity was detected at an apparent molecular weight of 60 kDa for pAKT as well as for AKT. As shown in Fig. 4 the pAKT and AKT expression is differently affected by AEZS-126 in the two cell lines. In parental A2780 cells the pAKT
level is clearly reduced by treatment with AEZS-126 (Fig. 4, lane 2 and 3) in contrast to untreated cells (Fig. 4, lane 1) and the expression level of AKT is nearly unaffected by AEZS-126 addition. In Acis2780 cells the amount of pAKT is also reduced by treatment with AEZS126 (Fig. 4, lane 5 and 6) compared to the untreated cells (Fig. 4, lane 4). But the amount of AKT is also dosedependently reduced in the cis-platinum resistant cells by treatment with AEZS-126 compared to untreated cells. To assess the mechanism of cell death, we incubated the ovarian cancer cell lines with increasing concentrations of AEZS-126 for 24 h and the markers of apoptosis and necrosis were analysed by fluorescence activated cell sorting (Fig. 5a, b). Early apoptotic cells are Annexin V-FITC positive and an increase in propidium iodide
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Fig. 3 Effects of AEZS-126 (a, b), rapamycin (c, d) and combination of both inhibitors (e, f) on the cell cycle distribution of A2780 (a, c, e) and Acis2780 (b, d, f) cells treated with the indicated amounts of
inhibitor or not for 24 h, fixed, permeabilized, stained with PI and analysed by flow cytometry. The figure shows the distribution of the cells to different phases of the cell cycle (%)
uptake precedes the loss of membrane integrity suggesting necrosis-like cell death. Annexin V-FITC and propidium iodide uptake would be suggestive of late apoptosis. AEZS-126 induced a dose-dependent increase of propidium iodide positive cell fraction in A2780 and Acis2780 cells suggesting an AEZS-126 induced loss of membrane integrity. There was no increase of Annexin V-FITC positive cells mediated by the incubation with AEZS-126 (Fig. 5a, b). Therefore it can be concluded that in both ovarian carcinoma cell lines only necrosis occurred and apoptosis is not induced by AEZS-126 in these cell lines. This observation fits well to the result of a cellular assay where the cell viability in both cell lines was not affected by co-incubation of AEZS-126 with the known inhibitor of apoptosis z-VAD-fmk (data not shown). In contrast incubation of A2780 and Acis2780 cells with increasing concentrations of AEZS-126 and different amount of
necrostatin-1 (75 and 100 lM), an inhibitor of necroptosis, resulted in increased cell viability (Fig. 5c, d). The cytotoxic effects of AEZS-126 were decreased by necrostain-1 in both ovarian cancer cell lines at 24 h of co-incubation with AEZS-126. Surprisingly the co-incubation of AEZS126 with the known inhibitor of necrosis Necrox-2 has no protective effect onto cell viability in these cell lines (Fig. 5e, f). Therefore it can be concluded that AEZS-126 induces only necroptosis—a form of programmed cell death with features of necrosis—in A2780 and Acis2780 cells. Subsequent to the proven growth inhibition of ovarian cancer cells by AEZS-126 and analysis of the molecular basis we focused next on the question if pre-treatment of platinum resistant cells with AEZS-126 resulted in an increased accessibility of these tumour cells for killing by NK-cells.
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Fig. 4 Western blotting showing expression of phosphorylated AKT1 (pAKT) and unphosphorylated AKT-1 (AKT) protein. The cells were incubated with the indicated amounts of AEZS-126 for 24 h and the protein expression was compared to untreated cells (control). In each lane 20 lg of total protein was analysed by 10 % SDS– polyacrylamide gel electrophoresis and transferred onto nitrocellulose. Membranes were probed overnight with anti-phospho-Akt
antibody or anti-Akt antibody (both from Epitomics, Burlingame, USA), then with a horseradish peroxidase-conjugated secondary antibody (Cell Signaling, Frankfurt, Germany). Antibodies were detected using chemiluminescent HRP substrate solution (Millipore, Schwalbach, Germany). The housekeeping protein b-actin was used as internal control
Therefore first of all the killing efficiency of NK-cells, prepared as described in the ‘‘Materials and methods’’, were proofed with a luciferase-based assay in the reporter cell line K562 (data not shown). After the proof of killing capacity, the NK-cells were used in a modified FATAL assay with A2780 and Acis2780 cells (Fig. 6). As shown in Fig. 6a the killing rate of tumour cells differ significantly between the two cell lines. Parental A2780 cells seem to be better targets for NK-cell mediated killing than the Acis2780 cells at all ratios between NK-cells and tumour cells used in this experiment. The same results were obtained with NK-cell preparations derived from other healthy donors. Therefore the observed differences in the killing rate between A2780 and Acis2780 cells are neither dependent on the healthy donor nor the NK-cell preparation. Pre-treatment of the tumour cells with AEZS-126 was performed for 12 h prior to co-culturing the ovarian cancer cells with the NK-cells. Such treatment with AEZS-126 resulted in an increase of tumour cell killing by NK-cells (Fig. 6b). The specificity of this effect is underlined by the dose dependent increase in killed cells during the co-culture of Acis2780 cells with NK-cells. Pre-treatment of Acis2780 cells with 800 nM AEZS-126 lead to nearly the same killing rate by NK-cells as it was observed in parental A2780 cells (Fig. 6b).
eventually develop secondary resistance to platinum based agents and compounds [4], we focused in this study on two cell lines with the same genetic background but difference in regard to platinum resistance. The efficiency of AEZS126 as a single agent and in combination with rapamycin was investigated in cis-platinum sensitive (A2780 cells) and cis-platinum resistant (Acis2780 cells) models of human ovarian cancer. In both cell lines coexist a PTEN loss-of-function mutation with a PIK3CA-activating mutation [16, 26] and therefore these both daughter cell lines (A2780 and Acis2780) represent that due to this mutations in the PI3K pathway is one of the main reason for PI3K activation in ovarian cancer patients [27]. AEZS-126 showed good anti-tumour activity in both in vitro models of ovarian cancer. Previously we have reported that AEZS-126 showed also good anti-tumour activity in in vitro models of triple-negative breast cancer as well as in MCF-7 cells [28]. In the present study we found that AEZS-126 executes its cytotoxicity in A2780 and Acis2780 cells only through necroptosis, a form of programmed cell death with features of necrosis. This observation is in good agreement with results from former studies where it was shown that AEZS-126 induced necroptosis in a broad panel of human breast cancer cell lines [28]. Nevertheless in the same study was demonstrated that the main mechanism of AEZS-126 mediated cytotoxicity must not always be based on necroptosis. In some breast cancer cell lines AEZS-126 induced caspase independent apoptosis beside necroptosis [28]. However, the mode of cell death seems to be a rather characteristic trait of treated cell line and not of the compound. Accordingly, in A2780 and Acis2780 cells which underwent a necroptotic cell death, cytotoxic effects of AEZS-126 could not be
Discussion As induction of the PI3K/AKT pathway is frequently observed in ovarian cancer [10], we evaluated the selective PI3K-inhibitor AEZS-126 in preclinical models of ovarian cancer. Based on the fact that virtually all patients
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Fig. 5 Effects of AEZS-126 on the cell distribution (in %) of A2780 (a) and Acis2780 (b) cells treated with the indicated amounts of inhibitor or not for 24 h, stained with Annexin V-FITC and PI. The markers of apoptosis and necrosis were analysed by flow cytometry. The obtained results were verified in cellular assays (c–f). Effects of
AEZS-126 in combination with necrostatin-1 (c, d) and necrox-2 (e, f), respectively, on proliferation of A2780 (c, e) and Acis2780 (d, f) cells for 24 h. The mean value of three independent measurements is shown
abrogated by a multi-caspase inhibitor or by a necrosis inhibitor. But cell viability was increased by co-incubation with a specific necroptosis inhibitor. In general, it is well established that two signalling pathways initiate classical apoptosis: one acts through intracellular Bcl-2 proteins, the other through cell-surface pro-apoptotic receptors [29]. Apoptosis can be engaged by a range of cellular insults and one of the major modes of action of chemotherapeutic drugs may be via the activation of apoptosis [30]. In recent years, the co-existence of various apoptotic ways of cell death as well as the existence of caspase independent apoptotic pathways has been described [31, 32]. The clonogenic survival of cancer cells most probably relies on the simultaneous blockade of both apoptotic and non-apoptotic cell death mechanisms [33]. Furthermore, a form of programmed cell death with features of necrosis was discovered [34] and referred to as
necroptosis, meaning a cellular mechanism of necrotic cell death induced by apoptotic stimuli such as TNFa, FasL, and Trail [35]. Although occurring under regulated conditions, necroptotic cell death is characterised by the same morphological features as classical necrosis, so far regarded as an unregulated process [36]. The initiation of necroptosis, by death receptors, such as the TNFa receptor requires the kinase activity of receptor-interacting protein 1 (RIP1) and RIP3, and its execution involves the active disintegration of mitochondrial, lysosomal and plasma membranes [37]. Necroptosis was found in diseases, such as ischaemic injury, neurodegeneration and viral infection [38]. Furthermore in the development of higher vertebrates necroptosis seems to be a back up mechanism when apoptosis is inhibited [39, 40]. Recently necroptosis has also been demonstrated in tumour cells subsequent to treatment with agents such as shikonin and thus it was proposed to bypass cancer drug
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A2780 Acis2780
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Fig. 6 A2780 and A2780cis cells (105 cells/well), respectively, were used as targets in a modified 5 h FATAL assay using various tumour cell: NK-cell ratios. This assay was performed with untreated tumour cells (a) or with tumour cells pre-treated with the indicated amount of AEZS-126 (b). Target cell lysis was determined by flow cytometric analysis. The percentage of tumour cell lysis was determined in relation to a control containing tumour cells with medium alone. A representative of three independent experiments is shown
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resistance by activating multiple death pathways using necroptosis inducing agents [41]. As survival of tumour cells in vivo is strongly influenced by immunological parameters, immunotherapeutic strategies appear promising. NK-cells are a critical component of the innate immune response against infectious pathogens and malignant transformation [42, 43]. NK-cells mediate this activity through the elaboration of various cytokines as well as through direct cytolytic activity. However, unlike adaptive immune cells, which utilise specific clonal recognition receptors, NK-cell activation depends on a complex balance between activating and inhibitory signals [44, 45]. Nevertheless, NK-cells play an important role in immune surveillance and coordinating responses of other immune cells. Most tumour cells express surface molecules that can be recognised by activating receptors on NK-cells [6]. The expression of these receptors make such cells susceptible to endogenous NK-cells, but malignant cells have developed mechanisms to evade innate immune surveillance [7–9]. In patients with cancer, it is presumed that tumour cells have developed mechanisms to suppress NKcell activation and resist lysis by endogenous NK-cells, but the molecular basis for target resistance is not well understood. Recently first steps in characterization of the molecular basis for these resistance mechanisms in ovarian cancer with different AKT expression level were done [19]. As expected the cis-platinum-resistant Acis2780 cells are less accessible for NK-cell mediated killing compared to the parental A2780 cells. This finding is in agreement with a recent report by Bellucci et al. [46]. Using a
1:2
1:5
lentiviral shRNA library targeting more than 1,000 human genes they identify 83 genes that promote target cell resistance to human NK-cell-mediated killing [46]. Many of the genes identified in this genetic screen belong to common signalling pathways including members of the AKT/PI3K-pathway such as PIK3CA and PIK3CB [41]. A deeper analysis for the observed differences in regard to the NK-cell mediated killing demonstrated that different mechanism seems to be involved in. Beside an increased expression of anti-apoptotic genes (especially ciap-1 and 2) also an increased expression of ligands for the NK-cell receptor NKG2D (e.g. MICA and MICB) were detected in the platinum-resistant cells compared to parental A2780 cells [19]. Previously it was demonstrated that PI3K/AKT pathway is involved in inducing MICA/B expression in breast cancer cells [47]. Taken in account that the Acis2780 cells also express lower level of TIMP-3 the inhibitor of MICA/B shedding and at the same time the proteases for shedding are expressed a net increase of soluble MICA/B is present in Acis2780 cell cultures [19]. It is well known that proteolytic cleaved MICA/B protects cell against NK mediated cell killing [19, 48, 49]. Therefore it was concluded that the increased amount of soluble MICA/B is responsible for the lower killing rate of platinum-resistant Acis2780 cells compared to their parental A2780 cells [19]. Recently Bellucci et al. [46] demonstrated that treatment of tumour cells with JAK inhibitors increased susceptibility to NK-cell activity. The authors conclude that common signalling pathways can regulate susceptibility of human tumour cells to killing by
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immunologic effector cells and that small molecule inhibitors of these kinases may have important immunologic effects in vivo [46]. In this study we prove this hypothesis and demonstrated that also inhibition of PI3K/ AKT pathway renders the platinum-resistant Acis2780 cells accessible for NK-cell mediated killing. This is to our knowledge the first proof that inhibition of PI3K/AKT pathway by a small molecule results in an increased killing of tumour cells by NK cells; that would say to re-establish the innate immune surveillance. In conclusion AEZS-126 demonstrated a highly efficient anti-tumour activity in in vitro models of ovarian cancer. Due to the good anti-tumour activity and the expected increase in NK-cell mediated killing even of platinum resistant tumour cells, AEZS-126 seems to be a promising candidate for clinical testing in ovarian cancer. Acknowledgments We appreciate the permission to use the INTAS ChemoStar Imager (Department of Microbiology, University of Wu¨rzburg). Therefore we thank especially Professor T. Rudel and Dr. B. Bergmann. This work was supported by ,,Interdisziplina¨res Zentrum fu¨r Klinische Forschung ‘‘(IZKF) Wu¨rzburg. Conflict of interest All participating authors declare that they have no conflict of interest.
References 1. Jemal A, Siegel R, Xu J, Ward E (2010) Cancer statistics 2010. CA Cancer J Clin 60:277–300 2. Waldmann A, Eisenmann N, Katalinic A (2013) Epidemiology of malignant cervical, corpus uteri and ovarian tumours—current data and epidemiological trends. Geburtshilfe Frauenheilkund 73:123–129 3. Pignata S, Cannella L, Leopardo D et al (2011) Chemotherapy in epithelial ovarian cancer. Cancer Lett 303:73–83 4. Gonzalez-Martin AJ (2004) Medical treatment of epithelial ovarian cancer. Expert Rev Anticancer Ther 4:1125–1143 5. Matsuo K, Lin YG, Roman LD, Sood AK (2010) Overcoming platinum resistance in ovarian carcinoma. Expert Opin Investig Drugs 19:1339–1354 6. Lanier LL (2005) NK cell recognition. Annu Rev Immunol 23:225–274 7. Dunn GP, Bruce AT, Ikeda H et al (2002) Cancer immunoediting: from immunosurveillance to tumor escape. Nat Immunol 3:991–998 8. Orr MT, Lanier LL (2010) Natural killer cell education and tolerance. Cell 142:847–856 9. Smyth MJ, Dunn GP, Schreiber RD (2006) Cancer immunosurveillance and immunoediting: the roles of immunity in suppressing tumor development and shaping tumor immunogenicity. Adv Immunol 90:1–50 10. Altomare DA, Wang HQ, Skele KL et al (2004) AKT and mTOR phosphorylation is frequently detected in ovarian cancer and can be targeted to disrupt ovarian tumor cell growth. Oncogene 23:5853–5857 11. Cheng JQ, Lindsley CW, Cheng GZ et al (2005) The Akt/PKB pathway: molecular target for cancer drug discovery. Oncogene 24:7482–7492
123
Arch Gynecol Obstet (2015) 291:131–141 12. Steelman LS, Chappell WH, Abrams SL et al (2011) Roles of the Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR pathways in controlling growth and sensitivity to therapy—implications for cancer and aging. Aging (Albany NY) 3:192–222 13. Levine DA, Bogomolniy F, Yee CJ et al (2005) Frequent mutation of the PIK3CA gene in ovarian and breast cancers. Clin Cancer Res 11:2875–2878 14. Benedetti V, Perego P, Luca Beretta G et al (2008) Modulation of survival pathways in ovarian carcinoma cell lines resistant to platinum compounds. Mol Cancer Ther 7:679–687 15. Engel JB, Schonhals T, Hausler S et al (2011) Induction of programmed cell death by inhibition of AKT with the alkylphosphocholine perifosine in in vitro models of platinum sensitive and resistant ovarian cancers. Arch Gynecol Obstet 283:603–610 16. Santiskulvong C, Konecny GE, Fekete M et al (2011) Dual targeting of phosphoinositide 3-kinase and mammalian target of rapamycin using NVP-BEZ235 as a novel therapeutic approach in human ovarian carcinoma. Clin Cancer Res 17:2373–2384 17. Westfall SD, Skinner MK (2005) Inhibition of phosphatidylinositol 3-kinase sensitizes ovarian cancer cells to carboplatin and allows adjunct chemotherapy treatment. Mol Cancer Ther 4:1764–1771 18. Hahne JC, Honig A, Meyer SR et al (2012) Downregulation of AKT reverses platinum resistance of human ovarian cancers in vitro. Oncol Rep 28:2023–2028 19. Hahne JC, Meyer SR, Gambaryan S et al (2013) Immune escape of AKT overexpressing ovarian cancer cells. Int J Oncol 42:1630–1635 20. Yuon TL, Cantley LC (2008) PI3K pathway alterations in cancer: variations on a theme. Oncogen 27:5497–5510 21. Willems L, Tamburini J, Chapuis N et al (2012) PI3K and mTOR signaling pathways in cancer: new data on targeted therapies. Curr Oncol Rep 14:129–138 22. Behrens BC, Hamilton TC, Masuda H et al (1987) Characterization of a cis-diamminedichloroplatinum(II)-resistant human ovarian cancer cell line and its use in evaluation of platinum analogues. Cancer Res 47:414–418 23. Campling BG, Pym J, Galbraith PR, Cole SP (1988) Use of the MTT assay for rapid determination of chemosensitivity of human leukemic blast cells. Leuk Res 12:823–831 24. Valiante NM, Rengaraju M, Trinchieri G (1992) Role of the production of natural killer cell stimulatory factor (NKSF/IL-12) in the ability of B cell lines to stimulate T and NK cell proliferation. Cell Immunol 145:187–198 25. Sheehy ME, McDermott AB, Furlan SN et al (2001) A novel technique for the fluorometric assessment of T lymphocytic antigen specific lysis. J Immunol Methods 249:99–110 26. Oda K, Stokoe D, Taketani Y et al (2005) High frequency of coexistent mutations of PIK3CA and PTEN genes in carcinoma. Cancer Res 65:10669–10673 27. Dobbin ZC, Landen CN (2013) The importance of the PI3K/ AKT/mTOR pathway in the progression of ovarian cancer. Int J Mol Sci 14:8213–8227 28. Hahne JC, Schmidt H, Meyer SR et al (2013) Anti-tumour activity of phosphoinositide-3-kinase antagonist AEZS 126 in models of triple-negative breast cancer. J Cancer Res Clin Oncol 139:905–914 29. Leist M, Ja¨a¨ttela¨ M (2001) Four death and a funeral: from caspases to alternative mechanisms. Nat Rev Mol Cell Biol 8:589–598 30. Leong CO, Vidnovic N, DeYoung MP et al (2007) The p63/p73 network mediates chemosensitivity to cisplatin in a biologically defined subset of primary breast cancers. J Clin Invest 117:1370–1380
Arch Gynecol Obstet (2015) 291:131–141 31. Li LY, Luo X, Wang X (2001) Endonuclease G is an apoptotic DNase when released from mitochondria. Nature 412:95–99 32. Lin NU, Claus E, Sohl J et al (2008) Sites of distant recurrence and clinical outcomes in patients with metastatic triple-negative breast cancer: high incidence of central nervous system metastases. Cancer 113:2638–2645 33. Makin G, Hickman JA (2000) Apoptosis and cancer chemotherapy. Cell Tissue Res 301:143–152 34. Miao B, Degterev A (2009) Methods to analyze cellular necroptosis. Methods Mol Biol 559:79–93 35. Morris GJ, Naidu S, Topham AK et al (2007) Differences in breast carcinoma characteristics in newly diagnosed AfricanAmerican and Caucasian patients: a single-institution compilation compared with the National Cancer Institute’s Surveillance, Epidemiology, and End Results database. Cancer 110:876–884 36. Moulder SL (2010) Does the PI3K pathway play a role in basal breast cancer? Clin Breast Cancer 3:66–71 37. Pal SK, Childs BH, Pegram M (2011) Triple negative breast cancer: unmet medical needs. Breast Cancer Res Treat 125:627–636 38. Perou CM, Sorlie T, Eisen MB et al (2000) Molecular portraits of human breast tumours. Nature 406:747–752 39. Rakha EA, Ellis IO (2009) Triple-negative/basal-like breast cancer: review. Pathology 41:40–47 40. Rakha EA, El-Sayed ME, Green AR et al (2007) Prognostic markers in triple-negative breast cancer. Cancer 109:25–32
141 41. Rozman-Pungercar J, Kopitar-Jerala N, Bogyo M et al (2003) Inhibition of papain-like cysteine proteases and legumain by caspase-specific inhibitors: when reaction mechanism is more important than specificity. Cell Death Differ 10:881–888 42. Caligiuri MA (2008) Human natural killer cells. Blood 112:461–469 43. Vivier E, Raulet DH, Moretta A et al (2011) Innate or adaptive immunity? The example of natural killer cells. Science 331:44–49 44. Lanier LL (2008) Up on the tightrope: natural killer cell activation and inhibition. Nat Immunol 9:495–502 45. Moretta L, Biassoni R, Bottino C et al (2000) Human NK-cell receptors. Immunol Today 21:420–422 46. Bellucci R, Nguyen HN, Martin A et al (2012) Tyrosine kinase pathways modulate tumor susceptibility to natural killer cells. J Clin Invest 122:2369–2383 47. Okita R, Mougiakakos D, Ando T et al (2012) HER2/HER3 signaling regulates NK cell-mediated cytotoxicity via MHC class I chain-related molecule A and B expression in human breast cancer cell lines. J Immunol 188:2136–2145 48. Boutet P, Agu¨era-Gonza´lez S, Atkinson S et al (2009) Cutting Edge: the metalloproteinase ADAM17/TNF-a-converting enzyme regulates proteolytic shedding of the MHC class I-related chain B protein. J Immunol 182:49–53 49. Waldhauer I, Goehlsdorf D, Gieseke F et al (2008) Tumorassociated MICA is shed by ADAM proteases. Cancer Res 68:6368–6376
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GYNECOLOGIC ONCOLOGY
Anti-tumour activity of phosphoinositide-3-kinase antagonist AEZS-126 in models of ovarian cancer Jens C. Hahne • Antje Kurz • Susanne R. Meyer Johannes Dietl • Jo¨rg B. Engel • Arnd Honig
•
Received: 3 March 2014 / Accepted: 21 July 2014 / Published online: 13 August 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract Purpose Platinum resistance is the most crucial problem for treatment of ovarian cancer. There is a clinical need for new treatment strategies which overcome platinum resistance. Recently high level of AKT was shown to be involved in platinum resistance and furthermore in resistance against Natural-killer (NK)-cell mediated killing in ovarian cancer. Methods Here, we investigate the ability of the PI3K/ AKT inhibitor AEZS-126 alone and in combination with rapamycin to selectively target ovarian cancer cell proliferation and survival in vitro by MTT-assays and FACS based analysis. Furthermore the mechanism of cytotoxicity is analysed by FACS based assays. The NK-killing efficiency of ovarian cancer cells with and without pre-treatment with AEZS-126 was analysed. Results AEZS-126 showed good anti-tumour activity in in vitro models of ovarian cancer. Main mechanism of cytotoxicity seems to be necroptosis which could be abrogated by co-incubation with necrostatin-1. Furthermore pre-treatment of platinum resistant cells with AEZS126 resulted in an increased accessibility of these tumour cells for killing by NK-cells.
J. C. Hahne and A. Kurz have contributed equally to this work. J. C. Hahne A. Kurz S. R. Meyer J. Dietl A. Honig (&) Department of Gynecology, University Hospital of Wu¨rzburg, Josef-Schneider-Str. 4, 97080 Wu¨rzburg, Germany e-mail: [email protected] J. C. Hahne e-mail: [email protected] J. B. Engel Department of Gynecology, University of Regensburg, Landshuter Str. 65, 93055 Regensburg, Germany
Conclusion We demonstrated the highly efficient antitumour activity of AEZS-126 in in vitro models of ovarian cancer. Due to the good anti-tumour activity and the expected increase in NK-cell mediated killing even of platinum resistant tumour cells, AEZS-126 seems to be a promising candidate for clinical testing in ovarian cancer. Keywords Ovarian cancer AKT Platinum-resistance NK-cells
Introduction Ovarian cancer is the most common cause of cancer death from gynecologic tumours and currently causes about 100,000 deaths per year world wide [1]. In 2009 a total of 7,430 women were diagnosed with ovarian cancer in Germany [2]. The relative 5-year survival for ovarian cancer is only 40 % in Germany [2]. While treatment of ovarian cancer with platinum-based agents has been established for decades, these still remain the most active substances for this entity [3]. Accordingly, primary resistance to platinum-based therapy is associated with a worse disease free and overall survival [4]. However, virtually all patients eventually develop secondary resistance to platinum based agents and compounds used for second or third line treatment. Other substances display substantially less anti-cancer activity as compared to platinum [5]. There is a definite clinical need to develop new treatment strategies to overcome platinum resistance. As survival is strongly influenced by immunological parameters, immunotherapeutic strategies appear promising; therefore a better understanding of the interaction between ovarian tumour cells and cells of the immune system is necessary.
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Natural-killer (NK)-cells play an important role in immune surveillance and coordinating responses of other immune cells. Most tumour cells express surface molecules that can be recognised by activating receptors on NK-cells [6]. The expression of these receptors make such cells susceptible to endogenous NK-cells, but malignant cells have developed mechanisms to evade innate immune surveillance [7–9]. In patients with cancer, it is presumed that tumour cells have developed mechanisms to suppress NKcell activation and resist lysis by endogenous NK-cells, but the molecular basis for tumour cell resistance against this lysis is not well understood. Alterations of the serine/threonine kinase AKT/PKB (protein kinase B) pathway have been detected in several human malignancies including ovarian cancer [9]. AKT has a broad range of downstream effectors that regulate cell processes such as cell growth, cell cycle progression, survival, migration, and angiogenesis [11]. The AKT pathway is a promising target for cancer therapy, as it is a main nodal point where extracellular and intracellular oncogenic signals are integrated. Due to the key role of AKT in malignant transformation numerous inhibitors of the AKTpathway have been developed, and are currently in various stages of clinical development [12]. In human specimens of ovarian cancer AKT was found to be activated in 68 % [10] and PI3K (phosphoinositide-3kinase), an upstream component of the AKT-pathway, was found to be mutated in 12 % of the cases [13]. Recent evidence by our group and others has shown that overactivation of the AKT-pathway may be associated with platinum resistance [14–19]. It was demonstrated that parental A2780 cells become platinum resistant by overexpression of AKT and that platinum resistance in Acis2780 cells can be overcome by transfection with siRNA downregulating AKT [18]. Furthermore recently it was shown that ovarian tumour cells with an elevated AKT expression levels are protected against NK-cell mediated killing [19]. Because of the well-known fact that combination therapy has the potential to overcome drug resistance or escape from oncogene addiction [16, 20] we evaluated in this work here not only the ability of the PI3K/AKT inhibitor AEZS-126 alone but also in combination with rapamycin to selectively target ovarian cancer cell proliferation and survival in vitro. The connections between the PI3K and mTOR (mammalian target of rapamycin) kinases are multiple and complex, including common substrates, negative feedback loops, or direct activation mechanisms [21]. The mammalian target of rapamycin (mTOR) and the PI3K signaling pathways are commonly deregulated in cancers and promote cellular growth, proliferation, and survival [21]. Second the possibility to recover the NK-cell recognition of cis-platinum resistant ovarian cancer cells by
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inhibition of the PI3K/AKT pathway in these tumour cells was analysed.
Materials and methods Reagents and cell lines AEZS-126 was kindly provided by Aeterna Zentaris GmbH (Frankfurt, Germany) and rapamycin was purchased from Sigma-Aldrich (St. Louis, MO, USA). A2780 and Acis2780 cell lines (both are p53 and KRAS wild-type cell lines) were obtained from ECACC (European Collection of Cell Cultures; Salisbury, UK). The cis-platinum-resistant Acis2780 cell line has been developed by chronic exposure of the parental cis-platinum-sensitive A2780 cell line to increasing concentrations of cis-platinum [22]. Cytotoxicity MTT assay To quantify the cytotoxicity of AEZS-126 the viability of cells was measured with a non-radioactive cell counting assay. Therefore cells were cultured in 96-well flat-bottom plates, in humidified 37 °C and 5 % CO2 atmosphere. The cell density was initially adjusted to 2 9 105 cells/ml in a final volume of 50 ll/well. Cells were treated with different concentrations of AEZS 126 as indicated for 24, 48 and 72 h respectively. For the last 4 h of incubation, cells were pulsed with 10 ll of tetrazolium salt [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT] labelling reagent (Roth, Karlsruhe, Germany) at a final concentration of 0.5 mg/ml. The colorimetric assay is based on the cleavage of yellow MTT to pure violet formazan crystals by metabolic active cells [23]. The crystals were solubilised by addition of 100 ll 10 % SDS in 0.01 M HCl to each well. Absorbance was measured spectrophotometrically using a 540-nm wavelength ELISA reader (Tecan, Ma¨nnedorf, Switzerland) and Magellan software. The experiments were performed in triplicates, and at least three independent experiments were performed for each cell line. Preparation of cell lysates and Western blotting Preparation of cell lysates was performed as previously described [18]. Membranes were probed overnight with anti-Phospho-Akt-antibody and anti-Akt-antibody from Epitomics (Burlingame, CA, USA) and anti-b-actin-antibody from Abcam (Cambridge, UK), respectively. Secondary horseradish peroxidase (HRP)-conjugated antibodies were from Cell Signaling (Frankfurt, Germany). The chemiluminescent HRP substrate solution (Millipore, Schwalbach, Germany) was used for detection.
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NK-cell preparation and lysis assay
in binding buffer at a cell density of 1 9 106 cells/ml. 5 ll of FITC-conjugated Annexin-V were added to 100 ll of the cell suspension and incubated 15 min at room temperature. Than the cells were washed with binding buffer and resuspended in 200 ll binding buffer. After addition of 5 ll propidium iodide (2 lg/ml) the cells were analysed by flow cytometry on a FACSCalibur (Becton–Dickinson, Heidelberg, Germany).
PBMC (primary blood mononuclear cells) were obtained from healthy volunteers by density gradient centrifugation (Biocoll; Biochrom AG, Berlin, Germany). Monocytes were depleted by adherence and the remaining nonadherent PBL were further cultured on irradiated (30 Gy) RPMI8866 feeder cells to obtain polyclonal NK-cell populations [24]. After 6 days of co-culture 500 U IL-2 (Interleukin-2; Peprotech, Hamburg, Germany) were added per ml and 48 h later the polyclonal NK-cell population (effector cells) was used in different killing assays. Therefore the NK-cells were labeled with eFluor 670 (eBioscience, Frankfurt, Germany) and lytic activity against CFSE (Carboxyfluoresceinsuccininidylester)stained (eBioscience, Frankfurt, Germany) tumour cells (targets; 105 cells/well) was assessed in a modified 5 h FATAL assay using various effector:target ratios [25]. Cells were detached by trypsinisation and target cell lysis was determined by flow cytometric analysis of 30,000 target cells in a FACScan flow cytometer (Calibur, BD Biosciences, Heidelberg, Germany). eFluor 670-negative target cells were selected by gating and the percentage of CFSE cells within this population was determined. Spontaneous leakage of CFSE was determined by incubating the target cells with medium alone. Flow cytometry For cell cycle analysis, cells were treated with AEZS-126 as indicated, harvested, fixed and permeabilized over night in ice-cold 70 % ethanol (Merck, Darmstadt, Germany). The cells were washed twice with PBS (phosphate buffered saline). RNA was digested with RNase A (Gibco Life Technologies, Paisley, UK). The DNA was stained with propidium iodide (50 lg/ml). Fluorescence was recorded in a FACSCalibur (Becton–Dickinson, Heidelberg, Germany). Instrument settings were adjusted to move the G0/ G1 peak to 200 relative fluorescence units. Cells to the left of this peak appeared to have DNA content below 2n, indicative of cell death. Aggregated cells were gated out. A total of 2 9 104 cells per condition were recorded.
Apoptosis assay Cellular apoptosis was measured by Annexin-V and propidium iodide staining using Annexin-V Apoptosis Detection Kit FITC (eBioscience, Frankfurt, Germany) according to the manufacturer’s protocol. Briefly, cells were treated with AEZS-126 as indicated, harvested, washed once with binding buffer (10 mM HEPES/NaOH, (pH 7.4), 140 mM NaCl, 2.5 mM CaCl2) and resuspended
Results First of all the capacity of AEZS-126 to inhibit the growth of human ovarian cancer cells was evaluated. Therefore A2780 and Acis2780 cells were chosen, because these cell lines have been used extensively in preclinical studies of ovarian cancer and have been shown to translate well to clinical results. Furthermore the cis-platinum resistant Acis2780 cells and the parental A2780 cells have the same genetic background but differ in regard to platinum resistance. The two human ovarian cancer cell lines were treated with increasing concentrations of PI3K inhibitor AEZS-126 or with solvent only for 24, 48 and 72 h. All experiments were repeated at least in triplicates. AEZS-126 displayed strong growth-inhibitory effects in both ovarian cancer cell lines that became more pronounced with increasing treatment time (Fig. 1a, b). The half maximal inhibitory concentration (IC50) values were approximately 3.5 lM for A2780 and 5 lM for Acis2780 after 24-hincubation. IC50 values at 48 and 72 h were approximately 2.5 lM for A2780 and 1 lM for Acis2780 (Fig. 1a, b). To analyse if AEZS-126 displays antiproliferative effects in metabolically inactive cells, we treated both cell lines with the compound while cultivating them in serumfree medium, i.e. medium devoid of growth factors. Increasing concentrations of AEZS-126 which ranged between 0.2 and 200 lM were used; however, no reduction of the cytotoxicity of the drug was observed (data not shown). Next the efficacy of rapamycin a well established mTOR inhibitor was evaluated. Both ovarian cancer cell lines were treated with increasing concentrations of rapamycin or with solvent only for 24, 48 and 72 h. All experiments were repeated at least in triplicates. As expected rapamycin displayed strong growth-inhibitory effects in both ovarian cancer cell lines that became more pronounced with increasing treatment time (Fig. 1c, d). IC50 values were approximately 13 lM for A2780 and 18 lM for Acis2780 after 24-h-incubation (Fig. 1c, d). IC50 values at 48 h were approximately 2.5 lM for A2780 and 0.5 lM for Acis2780. If cells were incubated for 72 h we found IC50 of approximately 0.08 lM for A2780 and 0.1 lM for Acis2780 (Fig. 1c, d).
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Fig. 1 Effects of AEZS-126 (a, b) and rapamycin (c, d) on proliferation of A2780 (a, c) and Acis2780 (b, d) cells for 24 h (black solid graph), 48 h (black dashed graph) and 72 h (grey graph). The mean value of three independent measurements is shown
As observed previously for AEZS-126 increasing concentrations of rapamycin which ranged between 0.1 and 20 lM also showed no antiproliferative effects in metabolically inactive cells (data not shown). Finally the parental A2780 and the cis-platinum resistant Acis2780 cells were treated with AEZS-126 and rapamycin in different combinations or with solvents only for 24, 48 and 72 h. All experiments were repeated at least in triplicates. The combination displayed strong growth-inhibitory effects in both ovarian cancer cell lines (Fig. 2). Adding rapamycin to AEZS-126 clearly increased its cytotoxic effect in all cell lines over the whole dose range. Adding rapamycin to AEZS-126 strongly increased its cytotoxic effect in A2780 cells (Fig. 2a, c, e), resulting in a growth inhibition by 20–40 % with rapamycin. In contrast in Acis2780 cells, addition of rapamycin to AEZS-126 resulted in a decreased viability of up to 60–90 %. These very strong effects were observed in the cis-platinum resistant cells by co-treatment with 0.2 lM AEZS-126 and 20 lM rapamycin at all time points (Fig. 2b, d, f). To assess whether AEZS-126 (Fig. 3a, b) and rapamycin (Fig. 3c, d) as single agent or in combination (Fig. 3e, f) arrested cell growth in a specific phase of the cell cycle or rather caused cell death in the ovarian cancer cell lines, we performed a flow cytometric DNA cell cycle analysis on inhibitor-treated A2780 (Fig. 3a, c, e) and Acis2780 cells
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(Fig. 3b, d, f). This revealed in A2780 cells for AEZS-126 a dose-dependent decrease of cells in the G0/G1 and G2/M phases of the cell cycle, whereas the fraction of hypodiploid cells appearing to the left of the G0 cell population was concomitantly increased (Fig. 3a). Since this subG0 population is indicative of cell death, it can be concluded that AEZS-126 not only arrests the growth of A2780 cells, but also actually kills A2780 tumour cells irrespective of their current state in the cell cycle. In contrast AEZS-126 induced in Acis2780 cells only a slight decrease in the G0/ G1 and G2/M phases of the cell cycle as well as a slight increase in subG0 population and S phase (Fig. 3b). This observation argues for cell growth arrest in G0/G1 and G2/ M phase of cell cycle. Rapamycin induced in both cell lines a strong increase in subG0 population and a decrease of cells in the G0/G1 and G2/M phases of the cell cycle (Fig. 3c, d). Co-incubation of the AEZS-126 concentrations representing IC50 for each cell line with increasing amounts of rapamycin resulted in both cell lines in an increased subG0 population (Fig. 3e, f). Whereas for the parental cell line A2780 a decrease of cells in the G0/G 1 and G2/M phases of the cell cycle can be observed, we found in the cis-platinum resistant cell line Acis2780 a decrease of cells in the G2/M phase of the cell cycle and an nearly unaffected amount of cells in G0/G1 phase. So it can be concluded that the combination of AEZS-126 and rapamycin actually kills A2780 cells irrespective of their
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current state in the cell cycle and in the case of Acis2780 cells the cells are arrested in the G0/G1 phase. Next we focused on the mode of action of AEZS-126 in both ovarian cancer cell lines. To enlighten the molecular mechanism of growth inhibition the presence of phosphorylated AKT (pAKT) and unphosphorylated AKT (AKT) were assessed by western blotting in both cell lines that had been treated with the indicated amounts of AEZS126 for 24 h (Fig. 4). AEZS-126 was added to every cell culture so that the concentrations represented 0.5 IC50 and IC50 for each cell line. Specific antibody reactivity was detected at an apparent molecular weight of 60 kDa for pAKT as well as for AKT. As shown in Fig. 4 the pAKT and AKT expression is differently affected by AEZS-126 in the two cell lines. In parental A2780 cells the pAKT
level is clearly reduced by treatment with AEZS-126 (Fig. 4, lane 2 and 3) in contrast to untreated cells (Fig. 4, lane 1) and the expression level of AKT is nearly unaffected by AEZS-126 addition. In Acis2780 cells the amount of pAKT is also reduced by treatment with AEZS126 (Fig. 4, lane 5 and 6) compared to the untreated cells (Fig. 4, lane 4). But the amount of AKT is also dosedependently reduced in the cis-platinum resistant cells by treatment with AEZS-126 compared to untreated cells. To assess the mechanism of cell death, we incubated the ovarian cancer cell lines with increasing concentrations of AEZS-126 for 24 h and the markers of apoptosis and necrosis were analysed by fluorescence activated cell sorting (Fig. 5a, b). Early apoptotic cells are Annexin V-FITC positive and an increase in propidium iodide
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inhibitor or not for 24 h, fixed, permeabilized, stained with PI and analysed by flow cytometry. The figure shows the distribution of the cells to different phases of the cell cycle (%)
uptake precedes the loss of membrane integrity suggesting necrosis-like cell death. Annexin V-FITC and propidium iodide uptake would be suggestive of late apoptosis. AEZS-126 induced a dose-dependent increase of propidium iodide positive cell fraction in A2780 and Acis2780 cells suggesting an AEZS-126 induced loss of membrane integrity. There was no increase of Annexin V-FITC positive cells mediated by the incubation with AEZS-126 (Fig. 5a, b). Therefore it can be concluded that in both ovarian carcinoma cell lines only necrosis occurred and apoptosis is not induced by AEZS-126 in these cell lines. This observation fits well to the result of a cellular assay where the cell viability in both cell lines was not affected by co-incubation of AEZS-126 with the known inhibitor of apoptosis z-VAD-fmk (data not shown). In contrast incubation of A2780 and Acis2780 cells with increasing concentrations of AEZS-126 and different amount of
necrostatin-1 (75 and 100 lM), an inhibitor of necroptosis, resulted in increased cell viability (Fig. 5c, d). The cytotoxic effects of AEZS-126 were decreased by necrostain-1 in both ovarian cancer cell lines at 24 h of co-incubation with AEZS-126. Surprisingly the co-incubation of AEZS126 with the known inhibitor of necrosis Necrox-2 has no protective effect onto cell viability in these cell lines (Fig. 5e, f). Therefore it can be concluded that AEZS-126 induces only necroptosis—a form of programmed cell death with features of necrosis—in A2780 and Acis2780 cells. Subsequent to the proven growth inhibition of ovarian cancer cells by AEZS-126 and analysis of the molecular basis we focused next on the question if pre-treatment of platinum resistant cells with AEZS-126 resulted in an increased accessibility of these tumour cells for killing by NK-cells.
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Fig. 4 Western blotting showing expression of phosphorylated AKT1 (pAKT) and unphosphorylated AKT-1 (AKT) protein. The cells were incubated with the indicated amounts of AEZS-126 for 24 h and the protein expression was compared to untreated cells (control). In each lane 20 lg of total protein was analysed by 10 % SDS– polyacrylamide gel electrophoresis and transferred onto nitrocellulose. Membranes were probed overnight with anti-phospho-Akt
antibody or anti-Akt antibody (both from Epitomics, Burlingame, USA), then with a horseradish peroxidase-conjugated secondary antibody (Cell Signaling, Frankfurt, Germany). Antibodies were detected using chemiluminescent HRP substrate solution (Millipore, Schwalbach, Germany). The housekeeping protein b-actin was used as internal control
Therefore first of all the killing efficiency of NK-cells, prepared as described in the ‘‘Materials and methods’’, were proofed with a luciferase-based assay in the reporter cell line K562 (data not shown). After the proof of killing capacity, the NK-cells were used in a modified FATAL assay with A2780 and Acis2780 cells (Fig. 6). As shown in Fig. 6a the killing rate of tumour cells differ significantly between the two cell lines. Parental A2780 cells seem to be better targets for NK-cell mediated killing than the Acis2780 cells at all ratios between NK-cells and tumour cells used in this experiment. The same results were obtained with NK-cell preparations derived from other healthy donors. Therefore the observed differences in the killing rate between A2780 and Acis2780 cells are neither dependent on the healthy donor nor the NK-cell preparation. Pre-treatment of the tumour cells with AEZS-126 was performed for 12 h prior to co-culturing the ovarian cancer cells with the NK-cells. Such treatment with AEZS-126 resulted in an increase of tumour cell killing by NK-cells (Fig. 6b). The specificity of this effect is underlined by the dose dependent increase in killed cells during the co-culture of Acis2780 cells with NK-cells. Pre-treatment of Acis2780 cells with 800 nM AEZS-126 lead to nearly the same killing rate by NK-cells as it was observed in parental A2780 cells (Fig. 6b).
eventually develop secondary resistance to platinum based agents and compounds [4], we focused in this study on two cell lines with the same genetic background but difference in regard to platinum resistance. The efficiency of AEZS126 as a single agent and in combination with rapamycin was investigated in cis-platinum sensitive (A2780 cells) and cis-platinum resistant (Acis2780 cells) models of human ovarian cancer. In both cell lines coexist a PTEN loss-of-function mutation with a PIK3CA-activating mutation [16, 26] and therefore these both daughter cell lines (A2780 and Acis2780) represent that due to this mutations in the PI3K pathway is one of the main reason for PI3K activation in ovarian cancer patients [27]. AEZS-126 showed good anti-tumour activity in both in vitro models of ovarian cancer. Previously we have reported that AEZS-126 showed also good anti-tumour activity in in vitro models of triple-negative breast cancer as well as in MCF-7 cells [28]. In the present study we found that AEZS-126 executes its cytotoxicity in A2780 and Acis2780 cells only through necroptosis, a form of programmed cell death with features of necrosis. This observation is in good agreement with results from former studies where it was shown that AEZS-126 induced necroptosis in a broad panel of human breast cancer cell lines [28]. Nevertheless in the same study was demonstrated that the main mechanism of AEZS-126 mediated cytotoxicity must not always be based on necroptosis. In some breast cancer cell lines AEZS-126 induced caspase independent apoptosis beside necroptosis [28]. However, the mode of cell death seems to be a rather characteristic trait of treated cell line and not of the compound. Accordingly, in A2780 and Acis2780 cells which underwent a necroptotic cell death, cytotoxic effects of AEZS-126 could not be
Discussion As induction of the PI3K/AKT pathway is frequently observed in ovarian cancer [10], we evaluated the selective PI3K-inhibitor AEZS-126 in preclinical models of ovarian cancer. Based on the fact that virtually all patients
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Fig. 5 Effects of AEZS-126 on the cell distribution (in %) of A2780 (a) and Acis2780 (b) cells treated with the indicated amounts of inhibitor or not for 24 h, stained with Annexin V-FITC and PI. The markers of apoptosis and necrosis were analysed by flow cytometry. The obtained results were verified in cellular assays (c–f). Effects of
AEZS-126 in combination with necrostatin-1 (c, d) and necrox-2 (e, f), respectively, on proliferation of A2780 (c, e) and Acis2780 (d, f) cells for 24 h. The mean value of three independent measurements is shown
abrogated by a multi-caspase inhibitor or by a necrosis inhibitor. But cell viability was increased by co-incubation with a specific necroptosis inhibitor. In general, it is well established that two signalling pathways initiate classical apoptosis: one acts through intracellular Bcl-2 proteins, the other through cell-surface pro-apoptotic receptors [29]. Apoptosis can be engaged by a range of cellular insults and one of the major modes of action of chemotherapeutic drugs may be via the activation of apoptosis [30]. In recent years, the co-existence of various apoptotic ways of cell death as well as the existence of caspase independent apoptotic pathways has been described [31, 32]. The clonogenic survival of cancer cells most probably relies on the simultaneous blockade of both apoptotic and non-apoptotic cell death mechanisms [33]. Furthermore, a form of programmed cell death with features of necrosis was discovered [34] and referred to as
necroptosis, meaning a cellular mechanism of necrotic cell death induced by apoptotic stimuli such as TNFa, FasL, and Trail [35]. Although occurring under regulated conditions, necroptotic cell death is characterised by the same morphological features as classical necrosis, so far regarded as an unregulated process [36]. The initiation of necroptosis, by death receptors, such as the TNFa receptor requires the kinase activity of receptor-interacting protein 1 (RIP1) and RIP3, and its execution involves the active disintegration of mitochondrial, lysosomal and plasma membranes [37]. Necroptosis was found in diseases, such as ischaemic injury, neurodegeneration and viral infection [38]. Furthermore in the development of higher vertebrates necroptosis seems to be a back up mechanism when apoptosis is inhibited [39, 40]. Recently necroptosis has also been demonstrated in tumour cells subsequent to treatment with agents such as shikonin and thus it was proposed to bypass cancer drug
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Fig. 6 A2780 and A2780cis cells (105 cells/well), respectively, were used as targets in a modified 5 h FATAL assay using various tumour cell: NK-cell ratios. This assay was performed with untreated tumour cells (a) or with tumour cells pre-treated with the indicated amount of AEZS-126 (b). Target cell lysis was determined by flow cytometric analysis. The percentage of tumour cell lysis was determined in relation to a control containing tumour cells with medium alone. A representative of three independent experiments is shown
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resistance by activating multiple death pathways using necroptosis inducing agents [41]. As survival of tumour cells in vivo is strongly influenced by immunological parameters, immunotherapeutic strategies appear promising. NK-cells are a critical component of the innate immune response against infectious pathogens and malignant transformation [42, 43]. NK-cells mediate this activity through the elaboration of various cytokines as well as through direct cytolytic activity. However, unlike adaptive immune cells, which utilise specific clonal recognition receptors, NK-cell activation depends on a complex balance between activating and inhibitory signals [44, 45]. Nevertheless, NK-cells play an important role in immune surveillance and coordinating responses of other immune cells. Most tumour cells express surface molecules that can be recognised by activating receptors on NK-cells [6]. The expression of these receptors make such cells susceptible to endogenous NK-cells, but malignant cells have developed mechanisms to evade innate immune surveillance [7–9]. In patients with cancer, it is presumed that tumour cells have developed mechanisms to suppress NKcell activation and resist lysis by endogenous NK-cells, but the molecular basis for target resistance is not well understood. Recently first steps in characterization of the molecular basis for these resistance mechanisms in ovarian cancer with different AKT expression level were done [19]. As expected the cis-platinum-resistant Acis2780 cells are less accessible for NK-cell mediated killing compared to the parental A2780 cells. This finding is in agreement with a recent report by Bellucci et al. [46]. Using a
1:2
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lentiviral shRNA library targeting more than 1,000 human genes they identify 83 genes that promote target cell resistance to human NK-cell-mediated killing [46]. Many of the genes identified in this genetic screen belong to common signalling pathways including members of the AKT/PI3K-pathway such as PIK3CA and PIK3CB [41]. A deeper analysis for the observed differences in regard to the NK-cell mediated killing demonstrated that different mechanism seems to be involved in. Beside an increased expression of anti-apoptotic genes (especially ciap-1 and 2) also an increased expression of ligands for the NK-cell receptor NKG2D (e.g. MICA and MICB) were detected in the platinum-resistant cells compared to parental A2780 cells [19]. Previously it was demonstrated that PI3K/AKT pathway is involved in inducing MICA/B expression in breast cancer cells [47]. Taken in account that the Acis2780 cells also express lower level of TIMP-3 the inhibitor of MICA/B shedding and at the same time the proteases for shedding are expressed a net increase of soluble MICA/B is present in Acis2780 cell cultures [19]. It is well known that proteolytic cleaved MICA/B protects cell against NK mediated cell killing [19, 48, 49]. Therefore it was concluded that the increased amount of soluble MICA/B is responsible for the lower killing rate of platinum-resistant Acis2780 cells compared to their parental A2780 cells [19]. Recently Bellucci et al. [46] demonstrated that treatment of tumour cells with JAK inhibitors increased susceptibility to NK-cell activity. The authors conclude that common signalling pathways can regulate susceptibility of human tumour cells to killing by
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immunologic effector cells and that small molecule inhibitors of these kinases may have important immunologic effects in vivo [46]. In this study we prove this hypothesis and demonstrated that also inhibition of PI3K/ AKT pathway renders the platinum-resistant Acis2780 cells accessible for NK-cell mediated killing. This is to our knowledge the first proof that inhibition of PI3K/AKT pathway by a small molecule results in an increased killing of tumour cells by NK cells; that would say to re-establish the innate immune surveillance. In conclusion AEZS-126 demonstrated a highly efficient anti-tumour activity in in vitro models of ovarian cancer. Due to the good anti-tumour activity and the expected increase in NK-cell mediated killing even of platinum resistant tumour cells, AEZS-126 seems to be a promising candidate for clinical testing in ovarian cancer. Acknowledgments We appreciate the permission to use the INTAS ChemoStar Imager (Department of Microbiology, University of Wu¨rzburg). Therefore we thank especially Professor T. Rudel and Dr. B. Bergmann. This work was supported by ,,Interdisziplina¨res Zentrum fu¨r Klinische Forschung ‘‘(IZKF) Wu¨rzburg. Conflict of interest All participating authors declare that they have no conflict of interest.
References 1. Jemal A, Siegel R, Xu J, Ward E (2010) Cancer statistics 2010. CA Cancer J Clin 60:277–300 2. Waldmann A, Eisenmann N, Katalinic A (2013) Epidemiology of malignant cervical, corpus uteri and ovarian tumours—current data and epidemiological trends. Geburtshilfe Frauenheilkund 73:123–129 3. Pignata S, Cannella L, Leopardo D et al (2011) Chemotherapy in epithelial ovarian cancer. Cancer Lett 303:73–83 4. Gonzalez-Martin AJ (2004) Medical treatment of epithelial ovarian cancer. Expert Rev Anticancer Ther 4:1125–1143 5. Matsuo K, Lin YG, Roman LD, Sood AK (2010) Overcoming platinum resistance in ovarian carcinoma. Expert Opin Investig Drugs 19:1339–1354 6. Lanier LL (2005) NK cell recognition. Annu Rev Immunol 23:225–274 7. Dunn GP, Bruce AT, Ikeda H et al (2002) Cancer immunoediting: from immunosurveillance to tumor escape. Nat Immunol 3:991–998 8. Orr MT, Lanier LL (2010) Natural killer cell education and tolerance. Cell 142:847–856 9. Smyth MJ, Dunn GP, Schreiber RD (2006) Cancer immunosurveillance and immunoediting: the roles of immunity in suppressing tumor development and shaping tumor immunogenicity. Adv Immunol 90:1–50 10. Altomare DA, Wang HQ, Skele KL et al (2004) AKT and mTOR phosphorylation is frequently detected in ovarian cancer and can be targeted to disrupt ovarian tumor cell growth. Oncogene 23:5853–5857 11. Cheng JQ, Lindsley CW, Cheng GZ et al (2005) The Akt/PKB pathway: molecular target for cancer drug discovery. Oncogene 24:7482–7492
123
Arch Gynecol Obstet (2015) 291:131–141 12. Steelman LS, Chappell WH, Abrams SL et al (2011) Roles of the Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR pathways in controlling growth and sensitivity to therapy—implications for cancer and aging. Aging (Albany NY) 3:192–222 13. Levine DA, Bogomolniy F, Yee CJ et al (2005) Frequent mutation of the PIK3CA gene in ovarian and breast cancers. Clin Cancer Res 11:2875–2878 14. Benedetti V, Perego P, Luca Beretta G et al (2008) Modulation of survival pathways in ovarian carcinoma cell lines resistant to platinum compounds. Mol Cancer Ther 7:679–687 15. Engel JB, Schonhals T, Hausler S et al (2011) Induction of programmed cell death by inhibition of AKT with the alkylphosphocholine perifosine in in vitro models of platinum sensitive and resistant ovarian cancers. Arch Gynecol Obstet 283:603–610 16. Santiskulvong C, Konecny GE, Fekete M et al (2011) Dual targeting of phosphoinositide 3-kinase and mammalian target of rapamycin using NVP-BEZ235 as a novel therapeutic approach in human ovarian carcinoma. Clin Cancer Res 17:2373–2384 17. Westfall SD, Skinner MK (2005) Inhibition of phosphatidylinositol 3-kinase sensitizes ovarian cancer cells to carboplatin and allows adjunct chemotherapy treatment. Mol Cancer Ther 4:1764–1771 18. Hahne JC, Honig A, Meyer SR et al (2012) Downregulation of AKT reverses platinum resistance of human ovarian cancers in vitro. Oncol Rep 28:2023–2028 19. Hahne JC, Meyer SR, Gambaryan S et al (2013) Immune escape of AKT overexpressing ovarian cancer cells. Int J Oncol 42:1630–1635 20. Yuon TL, Cantley LC (2008) PI3K pathway alterations in cancer: variations on a theme. Oncogen 27:5497–5510 21. Willems L, Tamburini J, Chapuis N et al (2012) PI3K and mTOR signaling pathways in cancer: new data on targeted therapies. Curr Oncol Rep 14:129–138 22. Behrens BC, Hamilton TC, Masuda H et al (1987) Characterization of a cis-diamminedichloroplatinum(II)-resistant human ovarian cancer cell line and its use in evaluation of platinum analogues. Cancer Res 47:414–418 23. Campling BG, Pym J, Galbraith PR, Cole SP (1988) Use of the MTT assay for rapid determination of chemosensitivity of human leukemic blast cells. Leuk Res 12:823–831 24. Valiante NM, Rengaraju M, Trinchieri G (1992) Role of the production of natural killer cell stimulatory factor (NKSF/IL-12) in the ability of B cell lines to stimulate T and NK cell proliferation. Cell Immunol 145:187–198 25. Sheehy ME, McDermott AB, Furlan SN et al (2001) A novel technique for the fluorometric assessment of T lymphocytic antigen specific lysis. J Immunol Methods 249:99–110 26. Oda K, Stokoe D, Taketani Y et al (2005) High frequency of coexistent mutations of PIK3CA and PTEN genes in carcinoma. Cancer Res 65:10669–10673 27. Dobbin ZC, Landen CN (2013) The importance of the PI3K/ AKT/mTOR pathway in the progression of ovarian cancer. Int J Mol Sci 14:8213–8227 28. Hahne JC, Schmidt H, Meyer SR et al (2013) Anti-tumour activity of phosphoinositide-3-kinase antagonist AEZS 126 in models of triple-negative breast cancer. J Cancer Res Clin Oncol 139:905–914 29. Leist M, Ja¨a¨ttela¨ M (2001) Four death and a funeral: from caspases to alternative mechanisms. Nat Rev Mol Cell Biol 8:589–598 30. Leong CO, Vidnovic N, DeYoung MP et al (2007) The p63/p73 network mediates chemosensitivity to cisplatin in a biologically defined subset of primary breast cancers. J Clin Invest 117:1370–1380
Arch Gynecol Obstet (2015) 291:131–141 31. Li LY, Luo X, Wang X (2001) Endonuclease G is an apoptotic DNase when released from mitochondria. Nature 412:95–99 32. Lin NU, Claus E, Sohl J et al (2008) Sites of distant recurrence and clinical outcomes in patients with metastatic triple-negative breast cancer: high incidence of central nervous system metastases. Cancer 113:2638–2645 33. Makin G, Hickman JA (2000) Apoptosis and cancer chemotherapy. Cell Tissue Res 301:143–152 34. Miao B, Degterev A (2009) Methods to analyze cellular necroptosis. Methods Mol Biol 559:79–93 35. Morris GJ, Naidu S, Topham AK et al (2007) Differences in breast carcinoma characteristics in newly diagnosed AfricanAmerican and Caucasian patients: a single-institution compilation compared with the National Cancer Institute’s Surveillance, Epidemiology, and End Results database. Cancer 110:876–884 36. Moulder SL (2010) Does the PI3K pathway play a role in basal breast cancer? Clin Breast Cancer 3:66–71 37. Pal SK, Childs BH, Pegram M (2011) Triple negative breast cancer: unmet medical needs. Breast Cancer Res Treat 125:627–636 38. Perou CM, Sorlie T, Eisen MB et al (2000) Molecular portraits of human breast tumours. Nature 406:747–752 39. Rakha EA, Ellis IO (2009) Triple-negative/basal-like breast cancer: review. Pathology 41:40–47 40. Rakha EA, El-Sayed ME, Green AR et al (2007) Prognostic markers in triple-negative breast cancer. Cancer 109:25–32
141 41. Rozman-Pungercar J, Kopitar-Jerala N, Bogyo M et al (2003) Inhibition of papain-like cysteine proteases and legumain by caspase-specific inhibitors: when reaction mechanism is more important than specificity. Cell Death Differ 10:881–888 42. Caligiuri MA (2008) Human natural killer cells. Blood 112:461–469 43. Vivier E, Raulet DH, Moretta A et al (2011) Innate or adaptive immunity? The example of natural killer cells. Science 331:44–49 44. Lanier LL (2008) Up on the tightrope: natural killer cell activation and inhibition. Nat Immunol 9:495–502 45. Moretta L, Biassoni R, Bottino C et al (2000) Human NK-cell receptors. Immunol Today 21:420–422 46. Bellucci R, Nguyen HN, Martin A et al (2012) Tyrosine kinase pathways modulate tumor susceptibility to natural killer cells. J Clin Invest 122:2369–2383 47. Okita R, Mougiakakos D, Ando T et al (2012) HER2/HER3 signaling regulates NK cell-mediated cytotoxicity via MHC class I chain-related molecule A and B expression in human breast cancer cell lines. J Immunol 188:2136–2145 48. Boutet P, Agu¨era-Gonza´lez S, Atkinson S et al (2009) Cutting Edge: the metalloproteinase ADAM17/TNF-a-converting enzyme regulates proteolytic shedding of the MHC class I-related chain B protein. J Immunol 182:49–53 49. Waldhauer I, Goehlsdorf D, Gieseke F et al (2008) Tumorassociated MICA is shed by ADAM proteases. Cancer Res 68:6368–6376
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