Novel smac mimetic APG-1387 elicits ovarian cancer cell killing through TNF-alpha, Ripoptosome and autophagy mediated cell death pathway
- Bao-Xia Li†1,
- Heng-Bang Wang†2, 3,
- Miao-Zhen Qiu†4,
- Qiu-Yun Luo1,
- Han-Jie Yi1,
- Xiang-Lei Yan1,
- Wen-Tao Pan1,
- Lu-Ping Yuan1,
- Yu-Xin Zhang1,
- Jian-Hua Xu2Email author,
- Lin Zhang5Email author and
- Da-Jun Yang1, 3Email author
© The Author(s). 2018
Received: 6 November 2017
Accepted: 8 February 2018
Published: 12 March 2018
The Correction to this article has been published in Journal of Experimental & Clinical Cancer Research 2018 37:108
Ovarian cancer is a deadly disease. Inhibitors of apoptosis proteins (IAPs) are key regulators of apoptosis and are frequently dysregulated in ovarian cancer. Overexpression of IAPs proteins has been correlated with tumorigenesis, treatment resistance and poor prognosis. Reinstalling functional cell death machinery by pharmacological inhibition of IAPs proteins may represent an attractive therapeutic strategy for treatment of ovarian cancer.
CCK-8 and colony formation assay was performed to examine cytotoxic activity. Apoptosis was analyzed by fluorescence microscopy, flow cytometry and TUNEL assay. Elisa assay was used to determine TNFα protein. Caspase activity assay was used for caspase activation evaluation. Immunoprecipitation and siRNA interference were carried out for functional analysis. Western blotting analysis were carried out to test protein expression. Ovarian cancer cell xenograft nude mice model was used for in vivo efficacy evaluation.
APG-1387 demonstrated potent inhibitory effect on ovarian cancer cell growth and clonogenic cell survival. APG-1387 induced RIP1- and TNFα-dependent apoptotic cell death in ovarian cancer through downregulation of IAPs proteins and induction of caspase-8/FADD/RIP1 complex, which drives caspase-8 activation. NF-κB signaling pathway was activated upon APG-1387 treatment and RIP1 contributed to NF-κB activation. APG-1387 induced cytoprotective autophagy while triggering apoptosis in ovarian cancer cells and inhibition of autophagy enhanced APG-1387-induced apoptotic cell death. APG-1387 exhibited potent antitumor activity against established human ovarian cancer xenografts.
Our results demonstrate that APG-1387 targets IAPs proteins to potently elicit apoptotic cell death in vitro and in vivo, and provide mechanistic and applicable rationale for future clinical evaluation of APG-1387 in ovarian cancer.
Ovarian cancer is the most lethal gynecological malignancy and the second most common gynecologic cancer in the world, with a high incidence of metastasis and recurrent rate [1, 2]. As one of gynecologic malignant tumors that do harm to women’s health, ovarian cancer can occur at any age. High recurrent rate and advanced stage at diagnosis are two critical challenge in the treatment of ovarian cancer [1, 3, 4]. The 5-year survival rate for ovarian cancer is only around 27% . New therapeutic strategies are urgently needed in the management of ovarian cancer . Despite advances intreatment strategy, many tumors are resistant to current therapeutic approaches due to defects in the apoptotic machinery of the cells . For this, mechanisms of apoptosis have become promising targets for therapy .
Apoptosis, also called programed cell death, includes the extrinsic (type 1) and intrinsic (type 2) cell death pathways. Most of the chemotherapies kill cancer cells via the intrinsic, mitochondrial mediated cell death pathway, while some stimuli such as in the immune/inflammatory responses, TNF-alpha, FAS ligand/TRAIL, can initiate extrinsic death signals from cell surface to downstream intracellular targets. This type 1 of cell death module activates caspase-8 through its cleavage, which can then activate effector caspases 3/7, or pro-death BH3-only protein Bid. The activated or truncated Bid (tBid) translocates to mitochondria and initiates type 2 cell death process.
Many efforts have been made to explore strategies to reactivate the apoptosis in cancer cells. This has led to the development of Smac mimetics, which are designed to neutralize inhibitor of apoptosis proteins(IAPs). The IAPs are a group of anti-apoptosis proteins including cellular-IAP1 (cIAP1), cellular-IAP2(cIAP2), X-linked inhibitor of apoptosis protein(XIAP). IAP proteins are over expressed in various human malignancies and are associated with treatment resistance, disease progression and poor prognosis . Smac has been found to be down-regulated in lung cancer, and decreased expression of Smac is associated with worse prognosis . IAPs exert their anti-apoptotic actions through direct inhibition of initiator and effector caspases. IAPs have also been shown to ubiquitinate caspase proteins, thereby indirectly inhibit apoptosis [11–14]. Recently, several antagonists of IAPs have been developed, including APG-1387, a Smac mimetic . APG-1387 and similar bivalent IAP antagonists have been shown to induce proteasomal degradation of IAPs, abrogate IAPs-mediated inhibition of caspases, and induce cell death [16, 17].
Autophagy is considered as a double-edged sword with regard to genesis, development and the treatment of tumors as it kills tumor cells but also protect tumor cells against injury . To date, no studies have confrmed the role of autophagy when treated ovarian cancer with APG-1387, and the association between autophagy and apoptosis remains unclear. Therefore, the present study was to investigate the effect of APG-1387 on viability, apoptosis, clonogenic survival and autophagy in SKOV3 and OVCAR3 ovarian cancer cell lines and analyzed the association between autophagy and apoptosis. By this, we tried to reveal the potential underlying regulatory mechanism of these processes.
Cell cultures and reagents
Human ovarian cancer cell lines SKOV3 and OVCAR3 were purchased from the American Type Culture Collection (ATCC) provided by Sparklebio. SKOV3 and OVCAR3 cells were maintained in RPMI medium 1640 (Gibco) supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA) and 1% penicillin/streptomycin. Cells were incubated in a 5% CO2 humidified incubator at 37 °C, and collected using 0.05% trypsin EDTA following the specified incubation period. The following primary antibodies were used: P62(#8025), phospho-H2AX(γ-H2AX;#9718), caspase-8(#9746), RIP1(#3493 s), Beclin1(3738 s), ATG7(#2631S), PARP (#9546S), caspase-3(#9665 s), cIAP1(#7065 s), cIAP2(3130 s), XIAP(#14334), FADD(#2782S), phospho-NF-κBp105/p50(4806S), NF-κB2p100/p52(#4882S), TNF-α (#6945s), TNF-α neutralizing antibody (7321s), and TNFR1(#3736S) were purchased from Cell Signaling Technology Inc.; GAPDH (#sc-47724) from Santa Cruz Biotechnology (SC); LC3 (#NB100–2220) from Novus Biologicals. Z-VAD-FMK (#V116) and Necrostatin-1(#N9037) were from Sigma. IKK-16(#S2882) from Selleck. The data were collected from at least three independent experiments.
The novel Smac mimetic, APG-1387 was provided by the Ascentage Pharma Group Corp. Limited (Taizhou, China). The storage concentration of APG-187 was 40 μM, stored at − 20 °C, and diluted in the corresponding culture medium just before use.
Cell viability assay
Cells were plated in 96-well plates at a density of 5 × 103 cells/ml. After 24 h, APG-1387 was added at different concentrations. Cells without APG-1387 treatment were the control group. Cells were incubated with various concentrations of drugs for 72 h. Cell viability was performed with the CCK-8 (Dojindo, Kumamoto, Japan) following the manufacturer’s instruction. The inhibition rate of cell proliferation was calculated for each well as (A450control cells – A450 treated cells)/A450control cells × 100%. Experiments were performed in triplicate. Cell viability was expressed as mean ± SD of absorbance from treated cells vs. control cells in triplicate.
Apoptosis analysis by Fluorescence microscopy
APG-1387-induced apoptosis was assessed by Hoechst33258 staining. Treated with 10 nM APG-1387 for 24 h, the cells were harvested and smeared on slides. The slides were air-dried, fixed in methanol-acetone (3/1, v/v), and stained with Hoechst33258 (5 μg/mL) at 37 °C for 20 min. Nuclear morphology was examined under fluorescence microscopy (DFC480; LeicaMicrosystems, Wetzlar, Germany) to identify cells undergoing apoptosis.
Apoptosis analysis by flow cytometry
Apoptosis was detected by an AnnexinV-propidium iodide (PI) apoptosis detection kit (4A Biotech Company Limited, FXP018–100; Beijing, China) as described previously . To determine the apoptotic rate, the cells were placed in 6-well plates and treated with APG-1387 (0, 10 nM, 30 nM) at a density of 2 × 105 cells/well for 24 h, and then collected. AnnexinV-FITC/PI (Becton Dickinson, USA) staining was performed, following the manufacturer’s protocol. The apoptotic rate was determined using Celluest software (FCM, Becton Dickinson, USA).
Colony formation assay
Colony formation assay was used to evaluate the effects of APG-1387 on the proliferation of ovarian cancer. The cells were cultured in 6-well microplates (300 cells/ well), Then cells were treated with indicated concentrations of APG-1387 in microplates for 7 days. Cells were stained with crystal violet (Sigma, St. Louis, Mo, USA) for 20 min. Images of the colonies were obtained using a digital camera (Canon, EOS350D, Tokyo, Japan).
ELISA Kit (Cusabio Biotech Co., Ltd., Wuhan, China) were used for TNF-alpha detection, according to manufacturer’s instructions. The absorbance was measured in an ELISA reader at 450 nm. The concentrations of cytokines were calculated according to the standard curve using each of the recombinant cytokines in the ELISA kits.
Cells were cultured in 10 cm plates, treated as indicated, washed twice with PBS, and harvested with 1 ml of the following lysis buffer: 20 mM TrisHCl (pH 7.5), 150 mM NaCl, 10% glycerol, 1% TritonX-100, 2 mM EDTA, with complete, EDTA-free protease inhibitor (Roche) and phosphatase inhibitors (Sigma). Cells were left on ice for 30 min and centrifuged at 12,000 rpm for 20 min. Three milligrams of protein lysate were used for immunoprecipitation using 2 mg of the antibodies overnight at 4 °C. The following day, 25 μl of protein A/G ultralink resin (Thermo Scientific) was added for 2 h at 4 °C. The IPs were washed three times with lysis buffer, then sample buffer was added and the beads were boiled for 5 min at 95 °C. The samples were then analyzed by SDS-PAGE followed by immunoblotting with the indicated antibodies.
Caspase activity assay
After the applied APG-1387 treatment, cytosolic proteins (30 mg pertreatment) were incubated with the caspase assay buffer, with corresponding caspase substrate (AcIETD-AFC for caspase-8 and Ac-DEVD-AFC for caspase-3). Caspase activity was performed with the caspase-8 Activity Assay Kit (Beyotime Biotechnology, Shanghai, China) and caspase-3 Activity Assay Kit (Beyotime Biotechnology, China) following the manufacturer’s specifications. The plates were analyzed on an automated microplate spectrophotometer (Thermo Molecular De-vices Co., Union City, USA) at 405 nm.
siRNA and GFP-LC3 interference
The target sequence for Beclin1-specific siRNAs were 5′-UGGAAUGGAAUGAGAUUAAT-3′and 5′-UGGAAUGGAAUGAGAUUAATT-3′, 5′-GCTGCCGTTATACTGTTCT-3′ another target sequences ATG7-specific siRNAs were 5′-GCCGUGGAAUUGAUGGUAUTT-3′, 5′-GAAGCUCCCAAGGACAUUATT-3′ and 5′-GGAUCCUGGACUCUCUAAATT-3′, for all of which and the control siRNA (no silencing) were synthesized by GenChem Co. (Shanghai, China). The plasmid GFP-LC3 was kindly provided by Beth Levine. Transfection was performed, following the manufacturer’s protocol.
Immunofluorescence staining was conducted using a procedure similar to that described previously . The cells were plated on sterile coverslips and treated with 0 nM (APG-1387 control group) and 3 nM APG-1387 for 24 h, and fixed with 4% paraformaldehyde (PFA) for 10 min at 37 °C. After fixation, a permeabilization step was conducted with 0.25% Triton-X 100 for 10 min at 4 °C, and the cells were subsequently incubated in blocking solution containing 4% bovine serum albumin (BSA) for 1 h at 37 °C. The nucleus was stained with DAPI (1 mg/ml) for 5 min at room temperature. Fluorescence images were then captured by a confocal laser scanning microscope (LSM 700) (Carl Zeiss, Oberkochen, Germany).
Western blot assays
Cells were treated with various concentrations of drugs for 24 h, harvested in lysis buffer and tumors were harvested in RIPA buffer. After incubating on ice for 30 min, cells were centrifuged at 12,000 g for 15 min at 4 °C, and supernatant was collected. Samples were then analyzed by western blot. Proteins were visualized by incubation with SuperSignal west pico reagents (NCI5079, Thermo), followed by exposure to radiograph film.
Nude mouse xenograft studies
Four-week-old BALB/c (athymic) nude mice were purchased from the Shanghai’SIPPR-BK laboratory animal Co., Ltd.. The animal study protocol was approved by institutional animal care and use committee (IACUC) at Ascentage. All animal experiments were performed at Ascentage. A total of 5 × 106 skov3 cells were subcutaneously injected into the right flank of nude mice. When tumor volume reached 100–150 mm3 on average, the mice were randomized into 6 treatment groups. APG-1387 or vehicle control was administered intravenously in a total volume of 200 μL at the indicated dosing schedules. Body weights and tumor volumes (V) were measured every 2 days. Tumor volumes were calculated according to the formula: V = (lenght × width2)/2.
Immunohistochemistry (IHC) staining
Xenografts were fixed, and embedded in paraffin; Tissue sections (4-mm) were blocked with 0.5% BSA. Cell apoptosis was detected by terminal deoxynucleotidyl transferased UTP nick end labeling (TUNEL) In Situ Cell Death Detection Kit (Roche), following the manufacturer’s specifications. We counted the number of positive cells for TUNEL in all areas of the section under a light microscope at × 20 magnification and calculated the number of TUNEL-positive cells per field.
All assays were performed in triplicate. Data are expressed as the mean ± SD. Statistical analyses were performed using an analysis of variance with SPSS 13.0 software. Statistical significance was set at two-sided P < 0.05.
Growth inhibition effects of APG-1387 on ovarian cancer
APG-1387 induces apoptosis and activates caspase-3/PARP in ovarian cancer
APG-1387-induced cell death is caspase-dependent
APG-1387 is RIP1-dependent in ovarian cancer induced apoptosis
APG-1387 induces apoptotic cell death through engagement of TNFR1 by TNF-alpha signaling pathway
APG-1387 induces autophagy in ovarian cancer
Inhibition of autophagy sensitizes ovarian cancer cells to A PG-1387-induced apoptosis
APG-1387 inhibits tumor growth as a single agent in xenografted ovarian models
Apoptosis plays an essential role for organ development, homeostasis, and immune defense and provides mechanisms for the anti-cancer therapies. In recent years, apoptosis has been widely studied in relation to the treatment of malignant tumors. Apoptosis may also be inhibited by a variety of proteins including members of the inhibitors of apoptosis (IAP) family . IAPs comprise a family of structurally similar proteins, such as cIAP1, cIAP2 and XIAP largely over expressed by most tumors. Restoring the apoptotic cell death machinery by pharmacological inhibition of IAPs proteins represents a compelling strategy for cancer therapy. APG-1387 is a novel Smac mimetic developed by Ascentage and currently being evaluated in phase I clinical trial. In our study, we investigated the in vitro and in vivo antitumor activity of APG-1387 in ovarian cancer. CCK-8 assay revealed that APG-1387 could significantly suppress the growth of ovarian cancer cells in a dose-dependent manner. Colony formation assay revealed that the number of cell clones decreased with increasing APG-1387 concentration, suggesting that the growth and viability of human ovarian cancer cells were largely inhibited by APG-1387. Our results showed that APG-1387 could effectively induce apoptosis in ovarian cancer. Previous studies have demonstrated that APG-1387 inhibits the growth and proliferation of NPC (nasopharyngeal carcinoma) cells in a dose-dependent manner . Moreover, morphological changes in apoptotic characteristics, such as cellular shrinkage, rounding, poor adherence, and round floating shapes in harmine-treated cells were also observed by fluorescence microscopy. Apoptosis was also detected through fluorescein AnnexinV-FITC/PI double labeling . Research indicates that APG-1387 induced cell apoptosis in a dose-dependent manner. DNA damage is an early event of apoptosis. Upon DNA damage, multiple events to promote cell survival and facilitate DNA repair . If damage is excessive, programmed cell death to eliminate the afflicted cell . APG-1387 treatment caused the formation of significant amounts of the phosphorylation of H2AX and increased apoptosis rates at concentration of 10 nM and 30 nM than the control group (P < 0.05). Two distinct apoptotic pathways have been identified: the intrinsic and extrinsic pathways . The intrinsic and extrinsic pathways converge by activating the effector caspases-3/PARP, ultimately leading to the fragmentation of DNA with resultant cell death . During apoptosis, caspase-3 is one of the key executioners of apoptosis in response to various stimuli  and play crucial roles in cell apoptosis . It has been determined that a variety of chemotherapeutic agents induce apoptosis through the activation of caspase-3 and PARP [32, 33]. The activated-caspase-3 and cleaved-PARP causes morphological and biological apoptotic changes . Our results revealed that APG-1387-induced apoptosis was related to down-regulation of IAPs expressions. The intrinsic and extrinsic pathways converge by activating the effector caspases-3/PARP. When APG-1387 was coadministered with or without Z-VAD-FMK, the growth inhibition of cell morphology and cell viability were partially blocked, and the activation of caspase-3 and PARP were also partially blocked. In previous studies, our team found that the in vitro antitumor effect of APG-1387 in NPC was RIPK1-dependent , caspase-8 is also involved in NF-κB signaling . For example, homodimers formed by caspase-8 are associated with activation of NF-κB by RIPK1 . Our study provided evidence that an RIP1-mediated apoptosis pathway could be activated in ovarian cancer cells. APG-1387 induced ovarian cancer cell death via decreasing expression of cIAP1, cIAP2, XIAP in a dose- and time-dependent manner. APG-1387 induced formation of a complex consisting of RIP1, FADD and caspase-8, resulting in apoptosis.
The recent trials of birinapant and DEBIO1143 (AT-406) reported patients showed a trend towards increased circulating TNF-α [15, 36]. Inaddition, the inhibition of NF-κB could sensitize the resistant to Smac mimetic/TNF-alpha treatment. TNF-alpha can increase the production of cytokines through binding to its receptors including TNF-alpha receptor 1 (TNFR1), which is ubiquitously expressed in human tissues and serves as a signaling receptor for TNF-alpha . The impacts of NF-κB are pervasive as its effects extend across multiple systems. Research within the last few years has revealed that members of the NF-κB transcription factor regulate cell viability by activating genes involved in intrinsic pathways . NF-κB1/p50 (p50, which is processed from p105), and NF-κB2/p52 (p52, which is processed from p100) make up the NF-κB family in mammals . Mechanistic studies revealed that the secretion of TNF-alpha after ovarian cancer cells were treated with APG-1387 and found that the secretion of TNF-alpha was in time-dependent manner. Treatment with APG-1387 significantly decreased the expression of NF-κB p50, NF-κB p52 proteins as well as RIP1 which were required for NF-kB activation.
Autophagy is a catabolic pathway conserved among eukaryotic cells . The conversion of LC3-I into LC3-II has been considered as a hallmark of autophagy . Beclin1, as a crucial regulatory protein, regulates autophagosome membrane formation [41, 42]. ATG7 are required for the initiation of autophagy, and mediate phagophore expansion and autophagosome formation . This process is initiated by formation of the phagophore, which expands and fuses to form a vehicle called an autophagosome. Autophagosomes eventually fuse with lysosomes, which degrade their contents. Therefore, autophagy allows cells to rapidly eliminate long-lived proteins and destructive organelles for energy recycling . It’s primarily a process for cell protection, playing a pivotal role in cell survival, differentiation, development, and homeostasis . In our study, we demonstrated that APG-1387 treatment increased the transition of LC3-I to LC3-II, increased the expression of Beclin1, decreased the expression of P62 and increased the accumulation of punctate LC3 in the cytoplasm in cells. Transfection of cells with Beclin1 siRNAs or ATG7 siRNAs blocked the accumulation of LC3-II after APG-1387 treatment, a result which indicating that APG-1387 induced the autophagy of ovarian cancer cells. In most cases, autophagy-associated death is accompanied with apoptosis . Autophagy is considered as a double-edged sword with regard to genesis, development and the treatment of tumors as it kills tumor cells but also protect tumor cells against injury . However, uncontrolled autophagy will gradually consume intracellular components and lead to cell death . In recent years, many studies have reported that autophagy plays an important role in tumorigenesis and is becoming a key regulator of cancer survival [48, 49]. While the term of autophagic cell death is still a matter of dispute, accumulating evidence suggested the possibility of treating malignant tumors through autophagic regulation. Drugs that potentially modulate autophagy are increasingly being used in clinical trials, and screens are being performed for new drugs that can modulate autophagy for therapeutic purposes . Cancer cells fight against external stimuli through autophagy, which is especially evident in solid tumor cells where blood supply is inadequate. Autophagy plays a role in promoting tumor cell growth and survival . Autophagy also protects certain tumor cells from radiation damage . It is speculated that the mechanism of this protective effect may be through autophagy to remove damaged macromolecules or mitochondria and other organelles, thereby protecting tumor cells from apoptotic apoptosis . Our results suggest that APG-1387 induces autophagy while triggering apoptosis. APG-1387-induced autophagy plays a role in protecting cell survival and inhibition of autophagy potentiates cytotoxicity of APG-1387 in ovarian cancer cells.
In vivo, APG-1387 potently inhibited SKOV3 xenograft growth in nude mice, without causing apparent toxicities. Therefore, APG-1387 is an efficient anti-ovarian cancer agent. These observations indicate that APG-1387 may be effective as a single agent in patients with ovarian cancer.
This research demonstrated that APG-1387 has a potent antitumor effect on ovarian cancer both in vitro and in vivo by inducing apoptosis. APG-1387 induced ovarian cancer cell death by modulating autophagy via regulating expression of IAP families. The preclinical activity and mechanism of APG-1387 in ovarian cancer indicated its potential as a novel therapeutic for the treatment of ovarian cancer.
This work was supported by National Natural Science Foundation of China (Grant number. 81602066 and 81772587); the Fundamental Research Funds for the Central Universities (Grant number. 16ykpy25); the third outstanding young talents training plan and clinical scientist plan of Sun Yat-sen University cancer center; Science and Technology Planning Project of Guangdong Province, China (Grant number. 2013A022100023);Science and Technology Planning Project of Guangzhou, China (Grant number. 201510161726583); the Project of International Science and Technology Cooperation of Fujian Province (Grant number. 2015I0002); and the Joint Funds for the innovation of science and Technology, Fujian province (Grant number. 2016Y91030065).
BXL, HBW, LZ, JHX and DJY conceived and designed the study. BXL and HBW performed the experiments. MZQ, QYL, HJY, XLY, WTP, LPY, and YXZ supported the experiments. BXL, HBW, JHX and DJY analyzed and interpreted the data. BXL and HBW wrote the manuscript. HBW and DJY revised the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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