The natural compound α-lipoic acid (LA) is a chiral fatty acid harboring a disulphide bond, which can be reduced to dihydrolipoic acid (DHLA) [1
]. The biologically active (R
)-enantiomer represents an essential co-factor in mitochondrial multi-enzyme complexes performing oxidative decarboxylation (alpha-ketoglutarate dehydrogenase and pyruvate dehydrogenase). Thus, it represents a crucial player in the citric acid cycle and aerobic metabolism. In addition, LA and DHLA form a potent redox couple displaying remarkable anti-oxidative properties—e.g., by chelating divalent metal ions and by regenerating vitamin C [2
]. LA was further shown to act anti-inflammatory by downregulating the pro-inflammatory NF-κB pathway [3
LA is not only de novo synthesized endogenously in mitochondria, but also occurs in animal sources such as meat and in vegetables, in which it is most often covalently bound and therefore negligible for dietary uptake [5
]. Marketed as antioxidant, LA is available as food supplement, and drug approval has been granted for treatments of chronic diseases associated with high levels of oxidative stress, such as diabetic polyneuropathy [8
Recently, studies have investigated the potential of LA and its derivative CPI-613 as candidate drugs in the treatment of various types of cancer in vitro and in vivo [9
]. Regarding LA, cytotoxic and tumor growth-inhibiting effects against a panel of different cancer cell lines have been demonstrated (colorectal, lung, breast, thyroid, skin) [10
]. Treatment with LA generally induces imbalance of reactive oxygen species (ROS) release with concomitant loss of mitochondrial membrane potential culminating predominantly in apoptotic cell death via the intrinsic mitochondrial pathway [15
], in which p53 was shown to be dispensable [15
]. Furthermore, the autophagic machinery is triggered upon treatment with LA as a means of cell survival [13
Previous studies indicated a putative synergistic action of LA and chemotherapeutic agents—e.g., 5-fluorouracil (5-FU) or etoposide—although the underlying mechanism remains to be elucidated since its multifaceted mode of action targets a plethora of cancer hallmarks [9
]. In addition, LA was shown to target O6
-methylguanine-DNA methyltransferase (MGMT), which is involved in the repair of alkylation DNA damage [20
], for its degradation and thereby increases the cytotoxic effects of the alkylating anticancer drug temozolomide [13
In healthy tissue, the tumor suppressor protein p53 is an important regulator of the DNA damage response (DDR), cell cycle progression, and apoptotic cell death [21
]. Upon DNA damage, p53 is able to activate the expression of DNA repair genes—e.g., GADD45A
, or MSH2
]—intervene in the cell cycle via p21
upregulation or causes transcription of pro-apoptotic genes such as BAX
, or NOXA
]. The p53 protein is tightly controlled by post-translational modifications such as ubiquitination and phosphorylation [25
], and is further modulated by the cellular redox state [26
]. Mutations of p53 in cancer cells lead to either inactivation (loss of function) or hyperactivation (gain of function), both of which are crucial alterations resulting in an abrogation of its tumor suppressive functionality [27
]. Colorectal cancer (CRC) is the third most frequently diagnosed cancer worldwide and 5-year-survival-rates are still devastating, stressing the need for improved therapy approaches [28
]. Interestingly, approximately 50% of all colorectal tumors bear p53 mutations, prevailing in distal and rectal tumors [28
]. Previous studies in different cancer cell lines indicated a differential p53 expression level upon LA treatment. On the one hand, depletion of p53 following LA treatment was observed [30
], while on the other hand phosphorylation of p53 without changes of the total p53 protein level [31
] or even a stabilization of p53 [19
] were reported.
Triggered by our observations that p53 is dispensable for LA-induced cytotoxicity in CRC cells and that LA induces degradation of the redox-sensitive MGMT protein, we aimed to shed light on the effects of LA on p53 in CRC. At first, we studied the impact of LA on p53 on protein and mRNA level in various CRC cell lines and assessed the p53 transcriptional response. Subsequently, the generation of ROS by LA and the influence of anti-oxidant supplementation on p53 depletion was evaluated. Next, the involvement of different pathways such as autophagy and the proteasomal degradation machinery as well as post-translational modifications were analyzed, making use of different pharmacological inhibitors and genetic means. Finally, we set out to evaluate putative synergistic effects of combining LA and antineoplastic drugs used in CRC and other malignancies.
2. Materials and Methods
R(+)-LA, chloroquine (CQ), N-Acetyl-Cysteine (NAC), and MG132 were purchased from Sigma (Deisenhofen, Germany). The anticancer drugs doxorubicin (Doxo) and 5-flurouracil (5-FU) were from Medac (Wedel, Germany) and provided by the pharmacy of the UMC Mainz. The Nrf2 inhibitor ML385 and curcumin were obtained from Hycultec GmbH (Beutelsbach, Germany) and MDM2 inhibitor Nutlin-3a was from Selleck Chemicals (Houston, TX, USA).
Primary antibodies included Hsp90α/β (F8, mouse monoclonal; Santa Cruz, no. sc-13119), p53 (DO-1, mouse monoclonal; Santa Cruz, no. sc-126), p53 (FL-393; rabbit polyclonal; Santa Cruz, no. sc-6243), p62 (mouse monoclonal; Santa Cruz, no. sc-28359), LC3B (rabbit monoclonal; Cell Signaling Technology, no. 3868), ATG5 (rabbit monoclonal, Cell Signaling Technology, no. 12994), ubiquitin (mouse monoclonal; Santa Cruz, no. sc-8017), Nrf2 antibody (rabbit monoclonal; GeneTex, no. GTX103322), MDM2 (mouse monoclonal; Santa Cruz, no. sc-56154), heme oxygenase-1 (HO-1; rabbit polyclonal; GeneTex, no. GTX101147), as well as p21 (C-19, rabbit polyclonal; Santa Cruz, no. sc-397). Monoclonal PARP-1 antibody was provided by Dr. Alexander Bürkle (University of Konstanz, Germany). Secondary antibodies conjugated with horseradish-peroxidase were purchased from Santa Cruz (anti-mouse) and Cell Signaling (anti-rabbit).
2.2. Cell Culture and Treatments
The human CRC cell line HCT116 and isogenic p53-deficient HCT116 cells were generously provided by Dr. Bert Vogelstein (John Hopkins University, Baltimore, USA). LS174 cells were a kind gift of Dr, Thomas Brunner (University of Konstanz, Konstanz, Germany). SW48, HT29, and RKO cells were provided by the Institute of Toxicology, University Medical Center Mainz. HCT116 and RKO were grown in DMEM, LS174T in IMDM, and HT29 as well as SW48 in RPMI1640 medium supplemented with 10% fetal calf serum and 1% penicillin/streptomycin at 37 °C in humidified atmosphere of 5% CO2. Media and supplements were obtained from Gibco Life Technologies (Darmstadt, Germany) or PanBiotech (Aidenbach, Germany). All cell lines were mycoplasma negative.
LA was prepared as a 200 mM stock solution in 100% ethanol and added to the cell culture medium as indicated. Ethanol served as solvent control (0 µM LA). NAC was directly dissolved in cell culture medium (5 mM). In combination studies, cells were pre-incubated with 5 mM NAC for 2 h prior to LA treatment. When CQ (100 mM in H2O) was used to block autophagy, CQ was added 16 h prior to harvesting of the cells in a final concentration of 20 µM. In experiments using MG132 (13 mM dissolved in DMSO), the proteasome-inhibitor was added 16 h after LA treatment using a dose of 10 µM. ML385 and Nutlin-3a were dissolved in DMSO and used at a final concentration of 5 µM. Inhibitors were added to cell culture medium 2 h prior and 24 h after LA-treatment.
Combination treatments with anticancer drugs (Doxo/5-FU) included 44 h of incubation with LA plus 4 h of treatment with Doxo/5-FU for western blot analysis and 48 h of incubation with LA plus 72 h of treatment with Doxo/5-FU for ATP assays and Annexin V/PI stainings.
2.3. Preparation of Protein Lysates and Cell Fractionation
Whole-cell extracts and cell fractionation was performed as described [13
]. After indicated time points, cells were harvested and whole cell lysis was performed. In short, cells were lyzed in buffer containing 25 mM Tris-HCl pH 8.0, 5 mM EDTA, 1 mM DTT, 0.5 M NaCl supplemented with complete protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Upon 15 min of incubation at 4 °C on a rotating platform, extracts were clarified by centrifugation (10 min, 10,000 rpm) and protein content was determined in a final step using Bradford assay.
Cell fractionation was conducted by cell lysis in buffer containing 10 mM HEPES-KOH pH 7.9, 1.5 mM MgCl2 and 10 mM KCl for 15 min on ice. The lysate was then supplemented with 10% NP-40 and vortexed for 30 s. After centrifugation, the cytoplasmic fraction was obtained in the supernatant. Cell pellets were washed using isotonic buffer (10 mM Tris-HCl pH 7.4, 150 mM NaCl) and nuclei were lyzed in buffer containing 25% glycerol, 20 mM HEPES-KOH pH 7.9, 420 mM NaCl, 1.5 mM MgCl2, 0.5 mM EDTA. Upon thorough resuspension and incubation for 20 min on ice, nuclear extracts were isolated by centrifugation. Protein content was determined using the Bradford assay.
Harvested cells were lyzed in buffer (40 mM Tris-HCl pH 8, 1 mM EDTA, 1 mM PMSF) containing 5 mM N-ethylmaleimide (NEM) for 10 min on ice. Afterwards, 10 mM cysteine was added in order to inhibit NEM and to avoid further alkylation of amine- and thiol-moieties and cells were sonicated. After centrifugation, equal amounts were saved for input analysis and remaining lysates were pre-clarified by incubation with Protein A/G PLUS agarose (Santa Cruz, Dallas, USA) for 2 h at 4 °C on a rotating platform. Incubation for 2 h at 4 °C with anti-p53 antibody (rabbit polyclonal) followed. Finally, antigen-antibody complexes were captured by incubation with Protein A/G agarose beads overnight at 4 °C. Upon thorough washing, beads were denatured at 95 °C for 5 min using non-reducing Laemmli buffer. Samples were analyzed by SDS-PAGE and western blot analysis as described below.
2.5. Transient Transfection with siRNA
Knockdown of ATG5 was performed using siGENOME SMARTpool siRNA purchased from Dharmacon (Lafayette, LA, USA). Non-sense, scrambled siRNA, also purchased from Dharmacon, was used as control. Transfections were carried out as reported previously [33
]. Briefly, cells were transfected with 10 nM siRNA using Lipofectamine RNAimax (Invitrogen, Darmstadt, Germany) for 24 h before LA treatment for 48 h. Knockdown of ATG5 was verified by western blot analysis.
2.6. SDS-PAGE and Immunoblot Analysis
Western blot analysis was performed as described [34
]. Equal protein amounts were separated by SDS-PAGE followed by transfer onto a nitrocellulose membrane (Perkin Elmer, Rodgau, Germany) with a wet blot chamber (BioRad, München, Germany). Afterwards, membranes were blocked with 5% nonfat dry milk in TBS/0.1% Tween-20 for 1 h at RT. Primary antibody incubation was performed overnight at 4 °C followed by 3 × 5 min washing in TBS/0.1% Tween-20. Membranes were incubated with appropriate secondary antibodies for at least 1 h at RT. After 3 × 5 min washing, proteins were detected using Western Lightning® Plus-ECL (Perkin Elmer, Rodgau, Germany).
2.7. Preparation of RNA and Quantitative Real Time PCR
Gene expression analysis was essentially performed as described previously [35
]. Total RNA was isolated using the NucleoSpin®
RNA Kit (Macherey-Nagel, Düren, Germany). RNA concentrations were determined using a NanoDropTM
2000 spectrophotometer (Thermo Scientific) and 0.5 µg of total RNA was transcribed into cDNA using the Verso cDNA synthesis Kit (Thermo Scientific, Dreieich, Germany). qPCR was performed with the SensiMixTM
SYBR Green & Fluorescein Kit (Bioline, London, UK) and the CFX96TM
Real-Time PCR Detection System (Biorad, München, Germany) with the primers specified below (Table 1
). In all three experiments, RT qPCR was conducted using technical duplicates. The analysis was performed using CFX ManagerTM
Software. Non-transcribed controls were included in each run. Finally, expression of genes of interest was normalized to GAPDH
. The solvent control was set to one.
2.8. Confocal Immunofluorescence Microscopy of p53
Immunofluorescence staining and confocal microscopy was conducted as described previously [36
]. Briefly, cells were seeded on cover slips and treated as indicated. Upon fixation with 4% paraformaldehyde, cells were washed with 100 mM glycine. After blocking using 5% bovine serum albumin (BSA) in PBS with 0.3% Triton X-100, samples were incubated with anti-p53 antibody (mouse monoclonal, 1:500 in blocking solution) for 1 h at RT. As secondary antibody goat-anti-mouse coupled with Alexa488 (1:400 in PBS plus 0.3% Triton X-100, 1 h at RT) was used. Nuclei were finally counterstained using TO-PRO-3 (1:100 in PBS). Cover slips were mounted using VectaShield (Vector Labs, Burlingame, USA) and fluorescence microscopy images were taken using a Zeiss AxioObserver Z1 microscope equipped with a confocal LSM710 laser-scanning unit (Zeiss, Oberkochen, Germany). Pictures were analyzed and processed using Image J.
2.9. Flow Cytometry-Based Analysis of Autophagy Induction
Using the CytoID®
Green Autophagy Detection Kit (Enzo Life Science, Lörrach, Germany), autophagy levels were monitored as reported [33
]. According to the manufacturer’s protocol, attached and detached cells were harvested, washed with PBS, and stained for 30 min at 37 °C in the dark in phenol-free medium with 0.1 % dye. After a final washing step, measurement of the samples was carried out using BD Canto II and gating was performed using FACSDiva software (BD Biosciences). Unstained samples were measured with each experiment to subtract autofluorescence.
2.10. Cell Death Measurement by FLOW cytometry
Cell death was measured by flow cytometry using AnnexinV/PI staining as previously described [15
]. In short, adherent and detached cells were harvested using Trypsin/EDTA, pelleted, washed with PBS, and resuspended in 50 µL binding buffer (10 mM HEPES pH 7.4, 140 mM NaCl, 2.5 mM CaCl2, 0.1% BSA) plus 2,5 µL AnnexinV-FITC (Miltenyi Biotec, Bergisch Gladbach, Germany). Upon 15 min incubation on ice, 430 µL binding buffer and 10 µL propidium iodide (PI; Sigma Aldrich, Munich, Germany; 50 µg/mL) were added and samples were analyzed using a BD Canto II (BD Biosciences, Heidelberg, Germany). Gating of living cells (Annexin V/PI double negative), early apoptotic cells (Annexin V-positive, PI-negative) [37
], and late apoptotic/necrotic cells (Annexin V/PI-double positive) [38
], as well as data evaluation was performed with BD Diva software.
2.11. Determination of Reactive Oxygen Species by Flow Cytometry
Levels of reactive oxygen species (ROS) were quantified using flow cytometry as described [39
]. Incubation with 400 µM H2
(Merck, Darmstadt, Germany) for 20 min in PBS served as positive control. Cells were loaded with 2.5 µM CM-H2
DCFDA (Invitrogen, Darmstadt, Germany) in PBS for 30 min at 37 °C in phenol red- and serum-free medium. Upon washing with PBS, cells were harvested using Trypsin/EDTA, pelleted, resuspended in PBS, and analyzed using a BD Canto II (BD Biosciences, Heidelberg, Germany) and evaluated using BD Diva software.
2.12. Assessment of Combination Effect
ATP assays were used to measure cell viability [15
]. According to the manufacturer’s protocol of CellTiter-Glo®
Luminescent Cell Viability Assay Kit (Promega, Mannheim, Germany), 0.5 × 10³ HCT116 cells per well were seeded in a white 96-well-plate. Drug doses were chosen on the basis of IC50
values and a constant ratio recommended for the Chou–Talalay-method [40
]. Upon measurement using the luminometer Fluoroskan Ascent FL (Thermo Scientific, Vantaa, Finland), data was processed and evaluated using CompuSyn (ComboSyn Inc., Paramus, NJ, USA) and combination indexes (CI) were calculated.
Experiments were performed independently three times, except when otherwise stated. Representative experiments are displayed. Values are presented as means + standard error of the means (SEM) using GraphPad Prism 7.0 software. Statistical analysis was performed using two-sided Student’s t-test and statistical significance was defined as p < 0.05.
The present work shows that the disulphide compound LA triggers ubiquitin-proteasome mediated degradation of p53 in various CRC cell lines and synergizes with DNA damaging antineoplastic drugs (doxorubicin, 5-FU), which usually cause stabilization of p53. First, we provide evidence that p53 is efficiently depleted by LA in a dose-dependent manner. Remarkably, this effect was not limited to wildtype p53, but also occurred in HT29 cells (Figure 1
) that express mutated p53 (R273H) [58
]. This is a hot spot mutation found within the central DNA binding domain (DBD) and confers a gain-of-function to the mutated p53 protein [59
]. The R273H mutant was reported to impair the activation of the transcription factor Nrf2 following oxidative stress, to promote cell survival, to facilitate cell invasion and cell migration [60
]. LA-triggered degradation of mutant p53, as observed in HT29 cells, might thus attenuate its pro-tumorigenic and metastatic activity.
Furthermore, we show that LA-dependent depletion of p53 was restricted to the protein level, whereas gene expression of p53
was only slightly affected. This finding is consistent with the notion that p53 is mainly regulated on the protein level by post-translational modifications, which can either stabilize p53 (phosphorylation, acetylation, etc.) or promote its degradation (ubiquitination) [53
]. Depletion of p53 on protein level was also reported in lymphoblastoid cell lines deficient or proficient for the DDR kinase ATM, at a similar dose-dependent manner starting with 250 µM LA after 24 h [30
]. Two other studies assessed the effects of LA on p53 phosphorylation on Ser-15, suggesting increased phosphorylation without changes in the total p53 protein level [31
]. Ser-15 phosphorylation of p53 is known to be catalyzed by apical DDR kinases, such as ATM, following DNA damage [33
]. However, no genotoxic effects of LA were observed in our previous study in HCT116 cells [15
]. In contrast to the aforementioned studies and our own data, another work reported an accumulation of p53 in HCT116 cells after 24 h of incubation with 50 µM LA [19
]. Unfortunately, the authors did not include dose–response experiments and were not able to detect a p53-dependent transcriptional response after LA exposure.
The depletion of p53 in CRC cells occurred already after 24 h in a dose-dependent manner and preceded the cytotoxic effects of LA, which became visible in LS174T and HT29 cells after 48 h (Figure 2
, Figure A1
). The genotoxic anticancer drug 5-FU caused a p53-dependent transcriptional response with upregulation of MDM2
and other p53-responsive genes, whereas this was not observed following LA treatment. The LA-mediated upregulation of p21
at high doses is consistent with our previous study, which showed that p21 induction by LA is independent of p53 [15
]. We further assessed cellular ROS formation and showed moderately increased ROS levels at 1000 µM LA, which is in agreement with the pro-oxidative effects of LA reported in several tumor cell lines [16
]. However, pre-treatment of cells with the thiol-donor and antioxidant NAC did not prevent LA-induced degradation of p53, but rather promoted this process. Whether or not NAC affects LA-triggered cytotoxicity is an open question, which has to be addressed in futures studies. In contrast, the polyphenol curcumin with a well-known antioxidative activity [44
] caused a moderate stabilization of p53 in HCT116 cells, which is consistent with a previous study [64
]. These findings indicate that the disulphide and/or its reduced dithiol group are the structural elements required for p53 degradation.
Subsequently, we analyzed the underlying cellular process responsible for p53 degradation in more detail. The ubiquitin-proteasome system and autophagy are the two major quality control pathways to maintain cellular homeostasis [65
]. As our previous study revealed autophagy induction by LA in HCT116 and RKO cells [13
], we monitored autophagosome formation in all CRC cell lines tested. Consistent with our previous finding, increased autophagosome formation was detected in HCT116 and RKO cells, while only weak autophagy induction was observed in LS174T and SW48 cells (Figure 4
). Nevertheless, the autophagy receptor p62 was strongly upregulated in all CRC cell lines. p62 is involved in selective autophagy via binding of ubiquitinated cargo molecules, which are then directed to the autophagosome for lysosomal degradation [66
]. The expression of p62 is upregulated by the transcription factor Nrf2, which is activated upon oxidative stress [67
]. Vice versa, p62 contributes to the activation of Nrf2 through inactivation of KEAP1, which targets Nrf2 for degradation under normal conditions [68
], thereby creating a p62-Nrf2 feedback loop. Multiple lines of evidence show that LA causes activation of the Nrf2 pathway and upregulation of Nrf2-dependent gene expression [69
]. The observed accumulation of p62 in CRC cells treated with LA could thus be attributable to the concomitant activation of Nrf2 by LA. Intriguingly, LA treatment caused a dose-dependent induction of Nrf2 and its downstream target HO-1 in CRC cells (Figure 5
). Another reason for increased p62 levels may be its co-aggregation with accumulated cargo molecules as described previously [68
]. However, neither pharmacological inhibition nor genetic abrogation of autophagy were able to rescue the LA-triggered depletion of p53 (Figure 4
), hence excluding a major role for autophagy in p53 degradation.
Apart from the abovementioned mutual regulation of Nrf2 and p62, Nrf2 activation can also increase proteolytic activity by upregulation of the 20S proteasome and the Pa28αβ (11 S) proteasome regulator, as demonstrated following oxidative stress and treatment with the Nrf2 inducers curcumin or LA [72
]. Consistently, LA-dependent Nrf2 activation was observed in CRC cells, which coincided with p53 depletion. However, pharmacological inhibition of Nrf2 by ML385 was not able to rescue p53 degradation (Figure 5
). It should be mentioned that the inhibitor itself had some effect on cell growth, indicating that a genetic approach may be more appropriate to elucidate the contribution of Nrf2 to the LA-triggered depletion of p53.
In contrast, co-immunoprecipitation studies and western blot analysis revealed increased ubiquitination and proteasomal degradation of p53 by LA treatment, which can be blocked by proteasome inhibition (Figure 6
). To figure out whether the E3 ubiquitin ligase MDM2 is responsible for the enhanced ubiquitination of p53, MDM2 levels were assessed on gene and protein level, showing no induction by LA. The pharmacological MDM2 inhibitor Nutlin-3a boosted p53 stabilization and the induction of the p53 targets p21 and MDM2, but did not abrogate the LA-mediated depletion of p53 and, concomitantly, p21 (Figure 7
). These results indicate that MDM2 is dispensable for p53 degradation triggered by LA. Nonetheless, to completely exclude a contribution of MDM2, a genetic approach is required since MDM2 was reported to harbor two different binding sites for p53. The N-terminal p53 binding domain is targeted by Nutlin-3a, thereby restoring the transcriptional response by p53. However, Nutlin-3a hardly inhibits p53 ubiquitination [73
], which is in line with the observed ubiquitinated p53 species in HCT116 cells after Nutlin-3a treatment (Figure 7
). The ubiquitination of p53 by MDM2 is likely mediated by a second binding site in the central part of MDM2 [73
]. Furthermore, it should be kept in mind that various other E3 ligases (e.g., Pirh2, COP1, Trim39, etc.) have been identified, which target p53 for ubiquitination and subsequent proteasomal degradation [74
]. Thus, it is also conceivable that one of those E3 ligases may be modulated by LA.
The molecular trigger of this enhanced p53 degradation following LA exposure likely involves chemical modification of critical thiol groups (cysteine, methionine) within the p53 protein. The central DBD harbors 10 cysteine residues, of which 6 display surface orientation and are thus prone to undergo modification, e.g. by oxidants or electrophiles [26
was shown in vitro to oxidize the cysteine residues in the p53 core domain, which are responsible for zinc coordination [75
]. Furthermore, oxidation of p53 destabilized p53 wild-type conformation and impaired its DNA binding in vitro, which could be reversed by the reducing agent DTT [76
]. More specifically, oxidation of Met340 located in the tetramerization domain was reported to disrupt p53 folding [77
Another possibility is the binding of LA as reactive disulphide to thiol-containing amino acids in p53, thereby destabilizing p53 conformation and facilitating its ubiquitination. Interestingly, the amino acids Cys182 and Cys277 present in the DBD of p53 are highly susceptible to chemical modification by the alkylating reagent N
]. In line with this finding, Cys residues in the proximal DBD including Cys182 were shown to be glutathionylated in tumor cells, which was further increased by anticancer drugs and oxidative stress [79
]. Intriguingly, the drug disulfiram (DSF), which also contains a reactive disulphide bond, also catalyzed p53 glutathionylation and direct modification of p53. This resulted in the ubiquitin-proteasome mediated degradation of p53 in HCT116 and HT29 cells [80
]. These findings are in striking conformance with our study, in which LA targeted p53 for enhanced degradation by the ubiquitin-proteasome machinery.
Finally, we demonstrate that LA attenuated the p53-dependent induction of the cell cycle regulator p21 after treatment with the genotoxic anticancer drugs 5-FU and doxorubicin. The combination regimen of LA and the anticancer drugs led to synergistic killing of CRC cells in a p53-dependent manner as determined by Chou–Talalay analysis (Figure 8
, Table 2
). Strikingly, this synergism disappeared when p53-deficient HCT116 cells were used. It is yet conceivable that also proteins other than p53 and p21 may contribute to the observed synergism. These results extend our previous study, which showed that LA potentiates the cytotoxicity of both 5-FU and the temozolomide in CRC cells [13
]. It is further remarkable that the combination of LA and doxorubicin provided the best synergistic effect both in viability studies and cell death measurements in CRC cells with p53 expression. In support of the data presented herein, LA increased apoptotic cell death induction by etoposide and ionizing radiation [19
]. LA was further reported to enhance the cytotoxicity of the antineoplastic drugs cis-Platin and etoposide in lung cancer cells [81
]. This body of evidence suggests that LA and its chemical derivatives [9
] are promising agents for targeting cancer cells.
In conclusion, our study showed for the first time that LA triggers the degradation of both wild-type and mutant p53 in a ubiquitin-proteasome dependent manner and elicits synergistic cell killing with genotoxic anticancer drugs in CRC cells by p53 degradation.