IAP antagonists Birinapant and AT-406 efficiently synergise with either TRAIL, BRAF, or BCL-2 inhibitors to sensitise BRAFV600E colorectal tumour cells to apoptosis
© The Author(s). 2016
Received: 21 April 2016
Accepted: 22 July 2016
Published: 12 August 2016
High expression levels of Inhibitors of Apoptosis Proteins (IAPs) have been correlated with poor cancer prognosis and block the cell death pathway by interfering with caspase activation. SMAC-mimetics are small-molecule inhibitors of IAPs that mimic the endogenous SMAC and promote the induction of cell death by neutralizing IAPs.
In this study, anti-tumour activity of new SMAC-mimetics Birinapant and AT-406 is evaluated against colorectal adenocarcinoma cells and IAP cross-talk with either oncogenic BRAF or BCL-2, or with the TRAIL are further exploited towards rational combined protocols.
It is shown that pre-treatment of SMAC-mimetics followed by their combined treatment with BRAF inhibitors can decrease cell viability, migration and can very efficiently sensitize colorectal tumour cells to apoptosis. Moreover, co-treatment of TRAIL with SMAC-mimetics can efficiently sensitize resistant tumour cells to apoptosis synergistically, as shown by median effect analysis. Finally, Birinapant and AT-406 can synergise with BCL-2 inhibitor ABT-199 to reduce viability of adenocarcinoma cells with high BCL-2 expression.
Proposed synergistic rational anticancer combined protocols of IAP antagonists Birinapant and AT-406 in 2D and 3D cultures can be later further exploited in vivo, from precision tumour biology to precision medical oncology.
KeywordsIAP antagonists TRAIL BRAF inhibitors BCL2 inhibitors Synergistic treatments Overcome resistance in colorectal cancer cells
Colorectal cancer (CRC) is one of the most common malignancies worldwide. Regarding pathophysiology, CRC development is linked to the acquisition of oncogenic mutations. In CRC, like in other tumour types, rational combination treatments can overcome resistance.
Inhibitors of Apoptosis Proteins (IAPs), also known as BIRCs (BIR domain containing proteins) are a class of highly conserved proteins characterized by the presence of Baculovirus IAP Repeat (BIR) domain, a Zn2+ ion coordinating protein–protein interaction motif , predominantly known for the regulation of caspases and immune signalling. These proteins are crucial for numerous cellular signalling networks. There are eight known mammalian IAPs/BIRCs; among them are cIAP-1, cIAP-2, XIAP and SURVIVIN . IAPs can be regulated by certain endogenous inhibitors of IAPs like the pro-apoptotic protein Second Mitochondria-Derived Activator of Caspases (SMAC/DIABLO) [2–4], a mitochondrial protein released into the cytosol during apoptotic induction .
Although several studies have demonstrated elevated levels of multiple IAPs in an array of human cancers, there have been opposing prognostic implications for IAPs in different tumour types, strongly suggesting that the role of IAPs in tumorigenesis is context-and cell type-dependent . Apart from apoptotic-caspase cascade, IAPs are essential modulators of innate immunity signalling, canonical and non-canonical NF-kB pathways as well as TGF-b signalling pathway . Elevated expression of IAPs in certain tumour types has been correlated with tumour survival and resistance to chemotherapy. So, a variety of anti-tumour therapeutics, especially small-molecule inhibitors against IAPs (IAP antagonist compounds (IAC), SMAC-mimetics) are being designed and clinically tested [7, 8]. Since targeting of IAPs can be only partially efficient as anti-cancer therapy, rational combination treatments with other targeted molecules against driver oncogenes, or apoptotic factors can be crucial to overcome tumour resistance.
BRAF is a proto-oncogene which encodes a serine/threonine kinase and regulates the MAP kinase/ERK signaling pathway, which is crucial for cell proliferation. Mutations in this gene have been associated with various cancers, including colorectal cancer (CRC) and display potent transforming activity associated with progression to metastasis . PLX4032 (Vemurafenib) is a BRAF inhibitor which has demonstrated selectivity for the mutated BRAFV600E compared to non-mutated BRAF oncoprotein [10, 11]. PLX4032 was approved by the FDA for the treatment of BRAF-mutated metastatic melanoma . Colorectal cancer and other cancer types show intrinsic resistance to BRAFV600E specific inhibitors, mainly due to feedback activation of EGFR. This is treated with combined treatments that include BRAF and EGFR inhibitors to achieve encouraging preclinical, as well as clinical results .
Exploitation of TRAIL apoptotic properties for cancer therapy has provided encouraging results in the last decade. TRAIL induces apoptosis via interacting with its death receptors DR4 and DR5, which in turn results in death-inducing signaling complex (DISC) formation and caspase-8 processing . Caspase-8 activation can then result in caspase-3 activation through the mitochondrial-independent pathway, and/or through the activation of Bid through the mitochondrial-dependent pathway . Moreover, many studies have shown that TRAIL is more efficient in induction of cancer cell death in combined treatments . In recent studies, 5-Fluorouracil displayed synergy with TRAIL in inducing apoptosis in mutant KRAS non-small cell lung carcinoma cells ; TRAIL-R2-specific antibodies and recombinant TRAIL can synergise to kill cancer cells .
Targeting BCL-2 anti-apoptotic complexes and pathways in cancer is a productive drug discovery and development field. The small molecule ABT-199, which antagonizes the activity of BCL-2, is one of the most promising examples being currently in clinical trials and shows activity in many lymphoid malignancies as a single agent and in combination with conventional chemotherapy agents [25, 26].
Apoptosis inhibition contributes to the survival and proliferation of tumors and plays an important role to current therapy resistance. Targeting apoptosis is therefore very promising for the development of new agents that may enhance current cancer therapies. Birinapant (TL32711), C42H56F2N8O6, is an antagonist of XIAP and cIAP1 with Kd value of 45 nM and <1 nM, respectively (Kd is the equilibrium constant involved in the dissociation of a compound into two or more compounds; the lower the Kd value the higher the affinity of the compound with the IAPs). Birinapant is a second-generation bivalent antagonist of IAP proteins that is currently undergoing clinical development for the treatment of cancer. It has been demonstrated, using a range of assays that evaluated cIAP1 stability and oligomeric state, that Birinapant stabilized the cIAP1-BUCR (BIR3-UBA-CARD-RING) dimer and promoted auto-ubiquitylation of cIAP1 in vitro, and this improved tolerability has allowed Birinapant to proceed into clinical studies . The pro-apoptotic effects of Birinapant on caspase-3 activation were evaluated in mice bearing 38C13 B-cell lymphoma, HCT116 colon carcinoma or MDA-MB-231 breast adenocarcinoma tumours .
AT-406 (SM-406), C32H43N5O4, is a novel and orally active antagonist of multiple IAP proteins (binds to XIAP, cIAP1 and cIAP2). This is the first SMAC-mimetic registered for clinical trials in patients with advanced cancer. Limited anti-tumour activity may suggest development rather as adjunct treatment . AT-406 acts as a strong radio sensitizer in human cervical cancer cells  and has demonstrated anti-ovarian cancer efficacy as a single agent and in combination with carboplatin . In addition, AT-406 is highly effective in induction of apoptosis in xenograft tumours and is currently in phase I clinical trials for the treatment of of solid and hematological human tumors .
In this study, we investigate the effect of IAPs inhibition by recently developed SMAC-mimetics Birinapant and AT-406 in colorectal tumour cells, their cross-talk with the TRAIL-induced apoptotic pathway, BRAF and BCL-2 oncogenic pathways and the underlying mechanisms that can efficiently overcome tumour resistance to apoptosis. Efficient protocols of inhibition of IAPs activity and anti-apoptotic effect are presented by using Birinapant or AT-406 alone and in their combinations with either TRAIL or with other inhibitors of pro-survival pathways, like BRAF-MEK and BCL-2. Synergistic rational anticancer combined protocols are presented depending on the tumour cell background, like resistance to individual treatments, BRAF mutation or BCL-2 overexpression. These can be later further exploited in vivo, thus validating a precision medicine approach.
DLD-1, HCT116, SW620, HT29, RKO, Colo-205 human colon adenocarcinoma and Caco-2 colon intermediate adenoma cell lines were obtained from American Type Culture Collection (ATCC). All cell lines used in this study were grown in D-MEM medium supplemented with 10 % Fetal Bovine Serum (#10270, ThermoFisher Scientific, Wlatham, MA, USA, antibiotics (penicillin/streptomycin) and amino acids. Cells were treated with the SMAC-mimetics Debio1143 (or AT-406) and TL32711 (or Birinapant, catalog No. S7015, Shelleck Chemicals, Europe) that block the interaction of IAPS with caspases. Cells were also treated with the BRAFV600E inhibitor PLX-4720 (catalog No. S1152, Shelleck Chemicals, Europe), the BCL-2 inhibitor ABT-199 (GDC-0199) (catalog No. S8048, Shelleck Chemicals, Europe) and TRAIL SuperKiller cc-TRAIL (ALX-522-020) (Alexis Biochemicals, Laussane, Switzerland).
Whole cell lysates were prepared with RIPA Buffer [50 mM Tris HCl pH: 8, 150 mM NaCl, 0.5 % sodium deoxycholate, 1 % NP-40, 10 % SDS]. Extracts were resolved on SDS-PAGE, and transferred to nitrocellulose membrane (Whatman, Scheicher & Schuell, Dassel, Germany). Membranes were incubated with the specific antibodies overnight at 4 °C, washed with TBS-Tween20 and incubated with the appropriate secondary antibody, for 1 h at room temperature. Antibodies were used against: XIAP (1:500-#610716, BD Biosciences, San Jose, CA, USA), cIAP-1 and cIAP-2 (1:750-/#sc-7943 and sc-7944, Santa Cruz Biotechnology, Heidelberg, Germany), DR4 and DR5 (1:5000-/#1139 and #2019, ProSci Incorporated, Poway, CA, USA),, FADD (1:1000-/#F36620, BD Biosciences, San Jose, California, USA), BID (1:500-/#sc-11423, Santa Cruz Biotechnology, Heidelberg, Germany), BAD (1:200-/#sc-941, Santa Cruz Biotechnology, Heidelberg, Germany), BAX (1:500-/#MS-711, ThermoFisher Scientific, Waltham, MA, USA), BCL-2 (1:200-/#sc-7382, Santa Cruz Biotechnology, Heidelberg, Germany), Tubulin (1:2000-#sc-8035, Santa Cruz Biotechnology, Heidelberg, Germany), Cleaved Caspase-3 (1:500-/#9661, Cell Signaling Technology, Danvers, MA, USA), Total Caspase-3 (1:1000-/#9662, Cell Signaling Technology, Danvers, MA, USA), PARP-1 (sc-7150, Santa Cruz Biotechnology, Heidelberg, Germany), RIP-1 (1:2000-/#610458, BD Biosciences, San Jose, CA, USA), RIP-3 (1:2000-/#NBP2-24588, Novus Biologicals, Littleton, CO, USA). Secondary antibodies were: Goat anti-mouse IgG-HRP Santa Cruz Biotechnology, sc-2005; a-rabbit IgG-HRP, Jackson, 111-035-003. All the antibodies were diluted in 5 % skim milk”. Antibody signal was obtained with the enhanced chemiluminescence plus Western blotting detection system (Amersham Biosciences, Uppsala, Sweden) after exposure to Kodak Super RX film. Values were measured using the Image-Quant software (Amersham Biosciences) and protein levels were normalized against housekeeping proteins (tubulin/GAPDH). Experiments were independently repeated three times and standard deviation is presented.
Two dimensional culture
For the 2D culture experiments, cells (5×103 cells/well) were grown on cover slips in 24-well plates in medium, at 37 °C. Photographs of the 2D cultures were taken under light and confocal microscope after the selected treatments and appropriate staining.
The nuclei were stained with Hoechst No. 33342 (catalog No. 62249, ThermoFisher Scientific, Waltham, MA, USA) for apoptosis detection; the cleaved caspase-3, a marker of apoptosis, was detected with cleaved caspase-3 antibody (#9661, Cell Signaling Technology, Danvers, MA, USA) and a fluorescent secondary antibody (Alexa Fluor 488, #A11008, ThermoFisher Scientific, Waltham, MA, USA)
Three dimensional culture
For three-dimensional culture Matrigel (catalog No. 356234, BD Biosciences, San Jose, CA, USA) was used. Poly-lysine pre-coated cover slips were put in 24-well plate. Matrigel was diluted with cold complete DMEM medium to a final concentration of 50 %, 200 μl were deposited on each well containing cover slips and the plate was incubated at 37 °C for 15’. 1-1.5×103 cells was diluted in 100 μL of cold DMEM and mixed with 100 μL of 100 % Matrigel. The resulting 200 μL were added to the wells with pre-warmed Matrigel. Plate was incubated in a humidified atmosphere at 37 °C with 5 % CO2 for 13–15 days. Photographs of the 3D cultures were taken under light and confocal microscope after the selected treatments and appropriate staining. The nuclei were stained with Hoechst No. 33342 (catalog No. 62249, ThermoFisher Scientific, Waltham, MA, USA) for apoptosis detection; the cleaved caspase-3, a marker of apoptosis, was detected with cleaved caspase-3 antibody (#9661, Cell Signaling Technology, Danvers, MA, USA) and a fluorescent secondary antibody (Alexa Fluor 488, #A11008, ThermoFisher Scientific, Waltham, MA, USA)
Cell viability assay
For growth studies the sulforhodamine B (SRB) (S1402-5G, Sigma-Aldrich, Taufkirchen Germany) assay was used. Firstly, tumour cells (5×103 cells/well) were seeded into 96-well micro titer plates and were allowed to attach overnight. Thereafter, the cell number in treated versus control wells was estimated after treatment with 10 % trichloroacetic acid and staining with 0.4 % SRB in 1 % acetic acid. The percentage of viable cell was plotted each time. SD was used for error bar generation. For the calculation of combined drug effects, the median effect analysis was used [27, 28]. Synergism was determined using the method previously described based on the Bliss Independence Model [28, 29].
Cell migration assay
Cells were trypsinised, washed with medium containing 1 % FBS, and counted. 105 cells were plated into upper chamber of an 8 μm-pore Transwell filter (#3422, Corning, NY, USA), mounted in a 24-well dish containing 10 % FBS medium. Filters were pre-coated with Fibronectin (f1141, Sigma-Aldrich, Taufkirchen, Germany). Cells were allowed to migrate at 37 oC, 5 % CO2 for 36–40 h, fixed with methanol and stained with 0.1 % w/v crystal violet. Underside of filters was observed with 40× objective and migrating cells were determined in ten randomly selected fields for each well. Experiments were performed in duplicate and repeated twice. For migration assay coupled with SMAC-mimetic, cells were treated with Birinapant for 24 or 48 h and then seeded on transwell in the presence of Birinapant and incubated at 37 °C for another 24 h.
RNA extraction, reverse transcription and real time-PCR
GAPDH: 5’-GAA GGT GAA GGT CGG AGT (FW)
5’-CAT GGG TGG AAT CAT ATT GGA (RV)
cIAP-1: 5’-TGT TGT CA ACTT CAG ATA CCA CTG G-3’ (FW)
5’-CAT CAT GAC AGC ATC TTC TGA AGA-3’ (RV)
cIAP-2: 5’-TCC GTC AAG TTC AAG CCA GTT-3’ (FW)
5’-TCT CCT GGG CTG TCT GAT GTG-3’ (RV)
XIAP: 5’-GAC AGT ATG CAA GAT GAG TCA AGT CA-3’ (FW)
5’-GCA AAG CTT CTC CTC TTG GAG-3’ (RV)
BIRC5: 5’-GCA CGG TGG CTT ACG CCT G-3’ (FW)
5’-AAC CGG ACG AAT GCT TTT TAT CC-3’ (RV)
DR4: 5’-TCC AGC AAA TGG TGC TGA C-3’ (FW)
5’-GAG TCA AGG GGC ACG ATG TT-3’ (RV)
DR5: 5’-CCA GCA AAT GAA GGT GAT CC-3’ (FW)
5’-GCA CCA AGT CTG CAA AGT CA-3’ (RV)
XIAP, DR4 and DR5 are overexpressed at the mRNA and protein levels in all adenocarcinoma cell lines. High levels of BCL-2 protein expressed in RKO cell line
Relative RNA levels of cIAP-1, cIAP-2, XIAP, SURVIVIN, DR4 and DR5 genes were evaluated by Real Time PCR analysis in seven colon cancer cell lines. Caco-2 is an intermediate adenoma cell line and was used as control. The other six cell lines are colorectal adenocarcinoma cell lines: DLD-1 and HCT116 bear KRAS and PI3KCA mutations, SW620 bears KRAS mutation, HT29 and RKO bear BRAF and PI3KCA mutations and Colo205 cell line bears BRAF mutation.
TRAIL receptors expression was evaluated by Q-PCR and western blot analysis: DR4 mRNA is mostly over-expressed in HCT116 and RKO (4-fold), as well as in SW620 and Colo205 (up to 3-fold) compared to Caco-2 respectively (Fig. 1a. lanes 3, 6, 4, and 7). On the other hand, protein levels of DR4 were found over-expressed in all cell lines. In HT29 cell line by 3.9 fold, in Colo205 by 3.2 fold, in RKO by 2.8 fold, in SW620 by 2.4 and in DLD-1 by 2.3 fold compared to Caco-2 intermediate adenoma cell line respectively (Fig. 1a, lanes 5, 7, 6, 4, 2). DR5 mRNA was also found over-expressed in all cell lines as compared to Caco-2, mostly in RKO (2.5-fold), as well as in HCT116 and SW620 (2-fold) cell lines compared to Caco-2 (Fig. 1a, lanes 6, 3, 4 respectively). Protein levels of DR5 were detected remarkably up-regulated in all cell lines compared to Caco-2: from 5.7-fold in HCT116 to 1.8-fold in RKO, as compared to Caco-2 cell line (Fig. 1a).
mRNA levels of cIAP-1 and cIAP-2 have no significant differences between Caco-2 and adenocarcinoma cell lines, while cIAP-2 protein was over-expressed in all cell lines compared to Caco-2 (Fig. 1a). mRNA level of SURVIVIN is overexpressed in all cell lines but mostly in HCT116 as compared to Caco-2 (Fig. 1a). On the other hand, most of examined BCL-2 family members involved in the intrinsic death pathway were found over-expressed in almost all adenocarcinoma cell lines utilized: BCL-2 protein in RKO cell line being the most upregulated, other overexpressed factors of this family are BAK and BAX (Fig. 1b).
Treatment with SMAC-mimetics Birinapant and AT-406 resulted in a decrease of cell viability and appearance of apoptotic characteristics in colorectal adenocarcinoma cells
Following the above findings regarding reduction of cell viability after Birinapant treatment, the effect of SMAC-mimetics treatments on apoptosis was tested. After 72 h treatment of RKO cells with 0.5–5 μM Birinapant, clear signs of apoptosis were detected, as shown by caspase-3 and PARP-1 cleavage (Fig. 2b, lanes 6, 7, 8) and by detection of fragmented nuclei in RKO cells (Fig. 2c, lower panel). On the other hand, AT-406 treatment in RKO cells resulted in a lower reduction of cell viability (15–20 %), as compared to Birinapant, while little effect on apoptotic markers like PARP-1 cleavage, caspase-3 cleavage (Fig. 2b, lanes 3–5), was detected, further confirmed by small number of crescent nuclei (Fig. 2c, middle panel). Parallel treatments of Birinapant and AT-406 in HCT116 cell line, resulted in no significant effects on either cell viability (Fig. 2a) or apoptotic markers (Fig. 2b and d), respectively
Birinapant treatment results in the reduction of cell migration properties of RKO cell line
The mild treatment effects of the tested SMAC-mimetics presented in the first part of this study, proposed for rational combined treatment protocols in resistant cell lines to overcome resistance. Co-treatments presented in the following second part of the study include rational combinations of SMAC mimetics with a) oncogenic BRAFV600E inhibitors, also not efficient as mono-treatments, b) other therapeutics targeting apoptosis, like TRAIL and c) BCL-2 inhibitors, in cells presenting high BCL-2 expression.
SMAC-mimetics synergise with BRAFV600E inhibitor towards efficient antitumour treatments in 3D cultures of colorectal adenocarcinoma cells
Treatment with SMAC-mimetics Birinapant and AT-406 can sensitize resistant-to-TRAIL cancer cells to apoptosis in 2D and 3D cell cultures
In order to provide further evidence of Birinapant-TRAIL efficiency, co-treatment protocols were tested in models involving tumour microenvironment, like 3D tumour cell cultures: a significant reduction of tumour size and disorganization (Fig. 5e) and higher staining of cleaved caspase-3, as shown in confocal microscopy images (Fig. 5f) was detected after the combined treatment of Birinapant and TRAIL for 6 days. Effects after mono-treatments of individual drugs were also detected.
ABT-199, a specific inhibitor of BCL-2, reduces cell viability and has synergistic effect with SMAC-mimetics Birinapant and AT-406 in RKO tumour cells
The present study investigates the efficiency of SMAC-mimetics Birinapant and AT-406 in a number of CRC cell lines. Oncogenic and apoptotic pathways are exploited towards establishing novel as well as efficient anti-cancer treatment protocols, which involve either Birinapant or AT-406 as single agents or their rational combinations with TRAIL, BRAF600E targeting drugs, and BCL-2 inhibitors.
Expression levels of IAPs, DRs and BCL-2 may guide for tailored targeted therapeutics
Analysis of expression levels of apoptotic factors in several colorectal (CRC) adenocarcinoma cell lines provides evidence for their potential importance as tumour markers and/or targets, complimenting previous reports. XIAP, DR4 and DR5 are overexpressed at the mRNA and protein levels in all adenocarcinoma cell lines. Notably, high levels of BCL-2 protein are detected in RKO adenocarcinoma cell line. This data were further exploited here towards developing rational and efficient preclinical protocols based on the oncogenic and apoptotic profile of the tumour cell lines.
Birinapant and AT-406 show a mild effect on CRC tumour cells as mono-treatments
Treatment of a panel of CRC cell lines with novel SMAC-mimetics Birinapant and AT-406 resulted in a decrease of cell viability and cell migration properties, as well as appearance of apoptotic characteristics of selected colorectal adenocarcinoma cells, like the aggressive RKO cells. Regarding cell migration, IAPs can directly control Rho GTPases, thus regulating cell shape and migration (1). Here, for the first time, reduction of cancer cell migration by Birinapant is reported.
This study provides further evidence and interest on Birinapant, a novel SMAC-mimetic and promising anticancer agent  and its recently published proof of mechanism data for quantifying apoptotic biomarkers in clinical trials . On the other hand, the mild treatment effects of the tested SMAC-mimetics propose for their rational combined treatment protocols in resistant cell lines, either with BRAFV600E inhibitors or with other therapeutics targeting apoptosis like TRAIL or BCL-2 inhibitors.
Combined treatments of SMAC mimetics with BRAF inhibitor sensitise resistant BRAFV600E Colorectal tumour cells
BRAFV600E Colorectal tumours show intrinsic resistance to BRAFV600E specific inhibitors Vemurafenib and Dabrafenib (13), otherwise very efficient against BRAFmut melanoma (12). Many studies, including the current, aim at improving the efficiency of Vemurafenib against colorectal tumours by rational combined treatments.
Here, SMAC-mimetics Birinapant and AT-406 can synergise with PLX4720, a Vemurafenib lead compound, towards efficient antitumour treatments of BRAFV600E colorectal adenocarcinoma cells in 2D and 3D cultures. It is of interest, that the successful protocol includes pretreatment with Birinapant with subsequent Birinapant- PLX4720 combined treatment, which is necessary for tumour cell death to be induced.
These results support the therapeutic combination of Birinapant with multiple chemotherapies, as shown for those therapies that can induce TNF secretion . Remarkably, recent studies have provided strong evidence that Birinapant co-treatment can overcome platinum resistance in a tumour-initiating subpopulation of ovarian cancer , as well as in a tumour-initiating AML subpopulation in combination with demethylating agents .
TRAIL can synergise with SMAC mimetics to efficiently drive resistant tumour cells to apoptosis
Despite the fact that during colorectal carcinogenesis a marked increase in sensitivity to TRAIL has been reported, colorectal adenocarcinomas like HT29 and RKO remain partially resistant to TRAIL-induced apoptosis . TRAIL resistance has been associated with defective ceramide signalling [38, 39]. Among the many studies of efficient combined treatments in colorectal cancer cells involving TRAIL: quercetin can enhance TRAIL-mediated apoptosis in colon cancer cells by inducing the accumulation of death receptors in lipid rafts  and the selective BRAF V600E inhibitor PLX4720 acts synergistically with TRAIL in order to overcome oncogenic PIK3CA resistance in colon cancer cells .
Here, treatment with SMAC-mimetics Birinapant and AT-406 can sensitize resistant-to-TRAIL and SMAC-mimetics HT29 cancer cells to apoptosis in 2D and 3D cultures. The combination of Birinapant and TRAIL leads to simultaneous activation of both the intrinsic and the extrinsic pathway. The same synergistic effect has been shown in the combined treatment of AT-406 and TRAIL. Inhibiting IAP function by SMAC-mimetics can apparently sensitise the HT29 TRAIL resistant cell line to TRAIL-Birinapant (or AT-406) combined treatments. These results are complementing those of other studies , where Birinapant in combination with TNF-a exhibits a strong anti-melanoma effect in vitro and enhances TRAIL potency in inflammatory breast cancer cells in an IAP-dependent and TNF-α-independent mechanism .
Combined targeting of apoptosis at the BCL-2 and IAPs level is efficient for CRC cells
Early in this study, expression analysis of apoptotic factors in the panel colon adenocarcinoma cell lines has provided evidence for a notable overexpression of BCL-2 in RKO cell line. Therefore treatments with ABT-199, a specific inhibitor of BCL-2 were performed, which resulted in reduction of cell viability in RKO cell line.
ABT-199 is shown here to act synergistic with SMAC-mimetics Birinapant and AT-406 on cell viability of RKO colon adenocarcinoma cells. ABT-199 selectively targets BCL-2 not BCL-XL and is active as a single agent in lymphoid malignancies such as CLL and non-Hodgkin lymphoma .
Recently, a very efficient synergistic protocol of BRAF with autophagy inhibitors in colorectal cancer cells has been presented, as another example of the advantages and better efficiency of rational combined treatments as compared to mono-treatments .
Efficient protocols of inhibition of IAPs activity and anti-apoptotic effect are presented by using Birinapant or AT-406 alone and in their combinations with either TRAIL (Figs. 5, 6 and 7) or with other inhibitors of pro-survival pathways, like BRAF-MEK (Fig. 4) and BCL-2 (Fig. 8). Synergistic rational anticancer combined protocols are presented depending on the tumour cell background, like resistance to individual treatments, BRAF mutation or BCL-2 overexpression. These rational combined treatments of agents targeting the tumour cell apoptotic machinery to restore some of its functions lost in cancer, can lead to efficient anti-cancer protocols, once further validated at the pre-clinical level and finally in the clinic.
BCL-2, B-cell lymphoma 2; BIRC, baculovirus IAP repeat; BRAF kinase, rapidly accelerated fibrosarcoma kinase; CRC, colorectal cancer; DISC, death-inducing signaling complex; IAPs, inhibitors of apoptosis proteins; PARP, poly (ADP-ribose) polymerase; RIP kinases, receptor-interacting protein kinases; SMAC, second mitochondria-derived activator of caspases; TRAIL, TNF-related apoptosis-inducing ligand
This work was supported by grants from the General Secretariat of Research and Technology of Greece (GSRT) SINERGASIA “THERACAN” 11SYN-1-485 and KRIPIS “STHENOS” MIS 447985.
Availability of data and materials
The dataset(s) supporting the conclusions of this article is(are) included within the article (Additional file 1).
PP and AP conceived the study and wrote the manuscript. PP, AG, AV and MP performed the experiments, analyzed and interpreted the data. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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