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The SW480 cell line is derived from a human colon adenocarcinoma, and SW620 cells are derived from a lymph node metastasis of the same patient. We have previously shown that lupulone induces apoptosis in SW480 cells, through a cross talk between the TRAIL-death receptor pathway and the mitochondrial apoptotic pathway. In SW620 cells, lupulone induced apoptosis only through TRAIL-death receptor activation. Both cell lines exhibit the same p53 mutations. Because p53 plays a central role in the response to cellular stresses by upregulating the transcription of several genes controlling apoptosis, we aimed to study the involvement of p53 on lupulone-triggered apoptosis. Our data show that in SW620 cells, lupulone upregulated p53 gene expression and caused a cloistering of p53 in the nucleus, allowing p53 to play a proapoptotic role by activating the TRAIL-death receptor pathway. In contrast, in lupulone-treated SW480 cells, p53 was translocated to the cytoplasm where it initiated a survival response associated with the up-regulation of antiapoptotic Bcl-2 and Mcl-1 proteins in an attempt to preserve mitochondrial integrity. These prosurvival effects of p53 in lupulone-treated SW480 cells were reversed by pifithrin-α, an inhibitor of p53 function, which caused a blocking of p53 in the nucleus leading to the down-regulation of Bcl-2 and Mcl-1, the up-regulation of proapoptotic Bax protein and TRAIL-death receptors leading to enhanced cell death. Our data support different functions of the same mutated p53 in colon adenocarcinoma and derived metastatic cells in response to the chemopreventive agent lupulone.
The p53 protein plays a central role in the response to a wide range of cellular stresses including DNA damage. Activated p53 leads to cell cycle arrest, cell senescence or apoptosis . This protein can act like a transcription factor and upregulate the transcription of several genes implicated in the control of cell proliferation or apoptosis . However, it was recently reported that the p53 protein was also able to modulate the intrinsic mitochondrial apoptotic pathway through interactions with members of the Bcl-2 family [3,4].
Most human cancers, including colon cancer, exhibitmutations of the p53 gene and altered protein function. The SW480 cell line is derived from a primary colon adenocarcinoma obtained from a 50-year-old white male, and the SW620 cell line is derived from a lymph node metastasis of the same patient. Therefore, these two related cell lines represent an interesting in vitro model of the progression of colon cancer from a primary tumor to its metastatic spread . Both cell lines exhibit a mutated p53 gene with a guanosine-to-adenosine mutation at codon 273, and a cytosine-to-thymidine mutation at codon 309 resulting, respectively, in arginine-to-histidine and proline-to-serine substitutions in the p53 protein . It was reported that despite these mutations, depending on the cellular stress, the p53 protein may retain its ability to induce DNA repair mechanisms, cell cycle arrest, and apoptosis [7,8].
Lupulone, a bitter acid of hops (Humulus lupulus L.), consists of a mixture of the isomers n-, co-, and ad-lupulones. We have previously shown that lupulone induces apoptosis in SW480 and SW620 cells by activating TRAIL-death receptor signaling pathways. In SW480 cells, lupulone induced a cross talk between the extrinsic and intrinsic apoptotic pathways involving mitochondrial alterations. In the metastatic SW620 cells, lupulone induced an activation of the extrinsic apoptotic pathway through TRAIL-death receptor activation . To gain more insight into the molecular mechanisms triggered by lupulone, particularly in TRAIL-resistant SW620 cells and the mitochondrial implication observed only in SW480 cells, we aimed to investigate the effects of lupulone on p53 functions in both cell lines. Indeed, activation of p53 may regulate DR4, DR5 TRAIL-death receptor, and/or Bcl-2 family member expression, such as Bcl-2 or Bax. Moreover, by transcription-independent mechanisms, the p53 protein may be translocated to the mitochondria, where it may interact with Bax, Bcl-2, or Bcl-XL and induce the release of cytochrome c [10,11].
Lupulone was obtained from an industrial by-product (Brasseries Kronenbourg, Strasbourg, France) and was isolated following the procedure described previously . Reverse transcription-polymerase chain reaction (RT-PCR) was performed by using different kits: RNeasy Mini kit, High-Capacity cDNA Archive kit, TaqMan Gene expression for primer of p53, TaqMan Universal PCR Master Mix, and ABI Prism 7500 Sequence Detection System, which were obtained from Applied Biosystems (Foster City, CA). Primary antibodies to p53, DR4, and DR5 were obtained from Alexis Biochemicals (Lausen, Switzerland). Primary antibodies to phospho-p53 (Ser-15) and phospho-p53 (Ser-392) were obtained from Abcam (Paris, France). An antibody to p53 conjugated with fluorescein isothiocyanate (FITC), which was used for p53 detection by flow cytometry, and the p53 chemical inhibitor pifithrin-α (PFT-α) were obtained from Calbiochem, Merck Chemicals (Darmstadt, Germany). Antibodies to Bcl-2 conjugated with FITC and primary antibodies to Mcl-1 and Bax were obtained from BD Biosciences (Erembodegem, Belgium).
SW480 and SW620 cells were obtained from the European Collection of Animals Cell Culture (Salisbury, UK). Cells were maintained in Dulbecco's modified Eagle medium containing 25 mM glucose and supplemented with 10% heat-inactivated (56°C) horse serum, 100U/ml penicillin, 100 µg/ml streptomycin, and 1% nonessential amino acids (Invitrogen Corp, Cergy Pontoise, France) and kept at 37°C in a humidified atmosphere with 5% CO2. For experiments after trypsinization (0.5% trypsin/2.6 mM ethylenediamine tetraacetic acid), cells were seeded at 1 x 106 cells in culture dishes (100-mm internal diameter). The culture medium was Dulbecco's modified Eagle medium supplemented with 3% heat-inactivated horse serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 5 µg/ml transferrin, 5 ng/ml selenium, 10 µg/ml insulin, and 1% nonessential amino acids (Invitrogen Corp).
To determine expression of p53 mRNA, extraction of total RNA and analysis by real-time RT-PCR were performed. Total RNA was extracted using the RNeasy Mini kit (QIAGEN, VWR, Copenhagen, Denmark) following the manufacturer's instructions. The High-Capacity cDNA Archive kit (Applied Biosystems) was used to reverse transcribe RNA (1 µg) in 20 µl of reaction mix, and the measurements of the transcription levels of the selected genes were performed with TaqMan Gene Expression assays (protein p53, Hs00153349; Applied Biosystems). β-Actin was assigned as an endogenous control (cat. no. Hs99999903; Applied Biosystems). Real-time RT-PCR was performed with TaqMan Universal PCR Master Mix and ABI Prism 7500 Sequence Detection System (Applied Biosystems Sequence detector; Applied Biosystems) in triplicate wells. Data were analyzed with a comparative threshold cycle (ΔCT) method. This method is used to determine the values of Δcycle threshold (ΔCt) by normalizing the average Ct value of each treatment with value of each opposite endogenous control (β-actin). Then, calculation of 2-ΔΔCt of each treatment and statistical analysis were performed as described by Livak and Schmittgen .
Cells were treated with lupulone (40 µg/ml) and harvested by trypsinization at 24 and 48 hours. Cell pellets were washed with phosphate-buffered saline (PBS) and were fixed with a solution of PBS containing 4% paraformaldehyde for 1 hour at 4°C in the dark. Cell pellets were washed with a solution of PBS/BSA 0.2%/Tween 0.5% and were incubated with FITC-conjugated mouse antihuman p53 antibody (Calbiochem) or FITC-conjugated mouse IgG1 monoclonal isotype control antibody (BD Biosciences) for 1 hour at 4°C in the dark. After washing with the solution of PBS/BSA 0.2%/Tween 0.5%, cells were resuspended in PBS (37°C), and the fluorescence (515 nm) of 10,000 events per sample was analyzed by FACScan and CellQuest Software (BD Biosciences).
Cells (8 x 106) were treated with PFT-α (30 µM) and/or lupulone (40 µg/ml) and harvested at 24 and 48 hours. Nuclear, mitochondrial, and cytosolic fractions were isolated from cells using the Nuclear Extract Kit and Mitochondrial Fractionation Kit (ActiveMotif Europe, Rixensart, Belgium), and the protein content of each fraction was measured with the Lowry method. Western blot analysis was performed as previously described  with a protein load of 50 µg.
SW480 or SW620 cells (1 x 106 cells) were seeded in culture dishes and pretreated with a p53 inhibitor, PFT-α (30 µM;Merck Chemicals) 1 hour before lupulone treatment (40 µg/ml). Cells were harvested by trypsinization at 24 and 48 hours, washed with PBS, centrifuged, and fixed with 1 ml methanol-PBS (9:1 vol./vol.) during a 1- to 3-hour incubation at -20°C. Cells were washed twice in PBS and resuspended in 200 µl of PBS containing 0.25 µg/ml RNase A and 0.1 mg/ml propidium iodide (Sigma Aldrich, Munchen, Germany). After incubating in the dark at 37°C for 30 minutes, the fluorescence of cells (10,000 events) was analyzed by flow cytometry using CellQuest software (FACScan; BD Biosciences).
Cells were pretreated for 1 hour with PFT-α at 30 µM and treated with lupulone (40 µg/ml) for 24 and 48 hours. After trypsinization, cell pellets were washed with PBS and incubated with monoclonal mouse antihuman antibodies to TRAIL-R1 (DR4; 1:100) or TRAIL-R2 (DR5; 1:100; Alexis Biochemicals, Lausen, Switzerland) for 30 minutes at 4°C. Cells were washed twice with PBS and incubated with FITC-conjugated goat antimouse IgG1 antibody (1:50; AbD Serotec, Düsseldorf, Germany) or with FITC-conjugated mouse IgG1 monoclonal isotype control antibody (1:50; BD Biosciences) for 30 minutes at 4°C in the dark. After washing with PBS, cells were resuspended in PBS and the fluorescence of 10,000 events per sample (515 nm) was analyzed by FACScan using CellQuest software (BD Biosciences).
SW480 cells were pretreated for 1 hour with PFT-α at 30 µM and treated with lupulone (40 µg/ml) for 24 and 48 hours. Cells were harvested by trypsinization and processed for the fixation (PBS/paraformaldehyde 4%) and permeabilization (PBS/BSA 0.2%/Tween 0.5%) steps. For Bcl-2 protein detection, cells were labeled directly with 20 µl of FITC-conjugated mouse antihuman Bcl-2 monoclonal antibody or FITC-conjugated mouse IgG1 monoclonal Isotype control antibody (BD Biosciences) for 30 minutes at 4°C in the dark. For Mcl-1 and Bax detection, cells were incubated with rabbit antihuman Bax polyclonal antibody or rabbit antihuman Mcl-1 polyclonal antibody (1:100; BD Biosciences) for 30 minutes at 4°C. After washing twice, FITC-conjugated swine antirabbit F(ab′)2 antibody was added (1:10; Abcam) for 30 minutes at 4°C in the dark. After washing twice in permeabilization buffer, the fluorescence of 10,000 cells was analyzed using a FACScan flow cytometer and CellQuest software (BD Biosciences).
All data were presented as mean ± SE from three independent experiments. Significant differences between control and treated groups were evaluated by one-way ANOVA analysis, Student's t-test, or the Student-Newman-Keuls multiple comparison test was used to determine the significance of differences between data.
We reported previously that lupulone activated apoptosis in SW480 and SW620 cells through TRAIL-death receptor signaling pathways. In addition, we showed a specific activation of the intrinsic mitochondrial pathway only in SW480 cells . To gain more insight into these processes, p53 mRNA and protein expression was assessed using real-time RT-PCR and flow cytometry.
The p53 mRNA expression increased significantly in SW620 cells after lupulone treatment in a time-dependent manner; a significant increase with time was observed in the amount of p53 transcripts (by three-fold at 24 hours and by eight-fold at 48 hours) compared with untreated control cells (Figure 1A). In lupulone-treated SW480 cells, the amount of p53 transcripts was significantly lower when compared with the level detected in SW620 cells and increased only marginally with time (by 1.5-fold at 24 hours and by 2-fold at 48 hours). However, a time-dependent increase in p53 protein expression was observed after lupulone treatment in both SW620 and SW480 cell lines (Figure 1B). These data suggested that in SW620 cells, p53 expression was mainly regulated at the transcriptional level, whereas in SW480 cells, the increase in p53 expression did not result from an up-regulation of p53 gene transcription but rather from posttranscriptional events. Nevertheless, it was clear that in both cell lines, lupulone was able to significantly enhance p53 protein expression.
Activation of the p53 protein is characterized by phosphorylation at specific sites, such as serine-15 or serine-392 [14,15]. Using Western blot analysis, we measured the amount of total p53 protein and phosphorylated p53 protein at serine-15 and serine-392 after 24 and 48 hours of lupulone treatment (Figure 2). In both cell lines, we observed a strong increase in p53 protein phosphorylation at serine-392 in a time-dependent manner, as well as an increase in the amount of total p53 protein (Figure 2). Concerning p53 protein phosphorylation at serine-15, lupulone induced an important increase after 48 hours of treatment in SW620 cells, but this effect was weak in SW480 cells.
Drugs may cause a stress signal favoring the activation and the translocation of p53 from the nucleus to the cytoplasm by transcription-independent mechanisms . UsingWestern blot analysis, we compared the amount of p53 present in the nucleus, cytosol, and mitochondria after lupulone treatment (Figure 3). After 48 hours of lupulone treatment, an increased amount of p53 was found in the nucleus of both cell lines (Figure 3A). However, an accumulation of p53 was observed in the cytosol and mitochondria of lupulone-treated SW480 cells, and such an effect was not found in SW620 cells where p53 was retained in the nucleus (Figure 3, B and C). These data indicate that in SW480 cells, but not in SW620 cells, lupulone treatment favored a translocation of p53 protein from the nucleus to the cytoplasm.
To determine the role of p53 in lupulone-triggered apoptosis, cells were treated with PFT-α (30 µM), a reversible inhibitor of p53-responsive genes which blocks p53-mediated apoptosis [16,17]. Cells were exposed to the inhibitor 1 hour before lupulone (40 µg/ml) treatment. The amount of dead or dying hypodiploid cells was determined with propidium iodide staining by flow cytometry, and these cells were detected in the sub-G0/G1 region [18,19]. In SW620 cells, PFT-α caused a significant (P < .05) diminution of cell death after lupulone treatment (Figure 4). Surprisingly, in lupulone-treated SW480 cells exposed to the p53 inhibitor, a huge increase in the amount of hypodiploid cells was observed. Indeed, after 48 hours of treatment with lupulone + PFT-α, the amount of cells in the sub-G0/G1 region was increased by 40% relative to SW480 cells treated with lupulone used as a single drug (Figure 4). Our data strongly suggested that p53 may act like a cell survival factor in lupulone-treated SW480 cells, and such an effect was not observed for the metastatic SW620 cells.
PFT-α inhibits p53 transcriptional activity and has been reported to block p53 translocation from nucleus to cytosol through a transcription-independent manner . Using Western blot analysis, we compared the amount of p53 present in nucleus and cytosol, after pretreatment with PFT-α (30 µM). In lupulone-treated SW480 cells, PFT-α blocked p53 in the nucleus and reduced the amount of p53 in the cytosolic fraction (Figure 5A). These data demonstrated that PFT-α hindered the translocation of p53 from the nucleus to the cytosol.
It has been reported that, by a transcription-independent manner and after translocation from nucleus to the cytosol or mitochondria, p53 can form heterocomplexes with Bcl-2 or Mcl-1 leading to the activation of Bax, inducing mitochondrial disruption [4,10]. Lupulone treatment induced a significant increase of Bax protein in a time-dependent manner (Figure 5B), leading to activation of the intrinsic apoptotic pathway through the disruption of the mitochondria . Here we show that p53 inhibition by PFT-α induced a further significant increase of Bax protein expression associated with a down-regulation of the Bcl-2 and Mcl-1 survival factors (Figure 5B).
Cell surface expression of TRAIL-DR4 and -DR5 death receptors was measured after inhibition of p53 functions by PFT-α in lupulone-treated SW620 and SW480 cells. Up-regulation of TRAIL-death receptors by p53, more specifically DR5 expression, has been reported during the activation of apoptosis [21,22]. In lupulone-treated SW620 cells, p53 inhibition by PFT-α caused a significant reduction in the expression of DR5 receptor, but DR4 receptor expression was only slightly affected (Figure 6A). These data suggested that in SW620 cells, p53 seemed to act as a proapoptotic regulator by enhancing DR5 expression in response to lupulone treatment. Moreover, the specific regulation of DR5 expression by p53 without affecting DR4 death receptor expression may explain the limited decrease of cell death observed after p53 inhibition. Conversely, in lupulone-treated SW480 cells, the inhibition of p53 by PFT-α induced a significant increase of both DR4 and DR5 expression (Figure 6B). These data also support the view that, in lupulone-treated SW480 cells, p53 plays the role of a cell survival factor.
After cells are exposed to drugs that initiate a stress signal, it has been reported that p53 activates different cellular responses, including DNA repair, cell cycle arrest, and/or apoptosis [23,24]. In both the SW480 cells obtained from a human colon adenocarcinoma and in the derived metastatic SW620 cells, p53 is mutated in the same position in codon 273 and codon 309, resulting in arginine-to-histidine and proline-toserine substitutions in the p53 protein . Despite these mutations, p53 can be activated in these cell lines and modulate cell growth or death [6,25]. In both SW480 and SW620 cells, we observed an upregulation of p53 mRNA and protein by lupulone when compared with untreated cells. However, the amount of p53 transcripts was far lower in SW480 cells compared with the amount detected in SW620 cells. In the latter, our data indicated that lupulone caused an up-regulation of p53 mainly at the transcriptional level, whereas in SW480 cells, the increase in p53 expression resulted mainly from a posttranscriptional regulatory mechanism. After lupulone treatment, p53 was phosphorylated at serine-392 in both cell lines, but the phosphorylation of serine-15 was specifically increased in SW620 cells and not in SW480 cells. Several studies have reported that phosphorylation at serine-15 prevents p53 from being exported from the nucleus and favors p53-controlled transcriptional activity [26,27]. Thus, the phosphorylation of p53 at serine-15 induced by lupulone in SW620 cells may favor the observed activation of the extrinsic apoptotic pathway through the previously reported  enhanced transcription of TRAIL-DR4 and -DR5 death receptors. In fact, our present data show that in the metastatic SW620 cells, p53 upregulated only the expression of the TRAIL-DR5 death receptor without affecting DR4 expression. This may explain why the inhibition of p53 by PFT-α, an inhibitor of p53-mediated transcription known to block p53-related apoptosis [28,29], caused only a moderate but significant (P < .05) reduction of SW620 cell death after lupulone treatment. Thus, in SW620 cells, p53 plays a proapoptotic role after lupulone treatment by regulating the expression of target genes, like DR5, resulting in an enhanced apoptotic response after lupulone treatment (Figure 7A).
In contrast to SW620 cells, the apoptotic responses of SW480 cells to lupulone are different and involved, as previously reported , a cross talk between the extrinsic (death receptor) and the intrinsic (mitochondrial) apoptotic pathways through Bid protein cleavage leading to mitochondrial disruption. In addition, we show here that in lupulone-treated SW480 cells, in contrast to what was observed in SW620 cells, p53 was translocated in the cytosol. It has been described that, by a transcription-independent manner, p53 can be translocated from the nucleus to the cytosol or mitochondria and can form protein heterocomplexes with Bcl-XL, Bcl-2, or Mcl-1 leading to the activation of Bax or Bad and, consequently, to mitochondrial disruption and release of cytochrome c to promote cell death [4,10,11,30]. Thus, the observed increase of p53 in the cytoplasm of lupulone-treated SW480 cells may participate in mitochondrial disruption. Surprisingly, after lupulone treatment, the inhibition of p53 function by PFT-α initiated increased cell death associated with an up-regulation of TRAIL DR4/DR5 death receptors and of the proapoptotic Bax protein concomitantly with the down-regulation of mitochondrial Bcl-2 and Mcl-1 survival factors. These data taken together showed that inhibition of p53 by PFT-α increases apoptosis induced by lupulone in SW480 cells. In these cells, p53 might upregulate the expression of genes whose products reduced the apoptotic signaling and favored cell survival. The inhibition of p53 function in lupulone-treated SW480 cells by PFT-α caused a blocking of p53 in the nucleus associated with a correlated reduced amount in the cytosol, confirming the already reported ability of PFT-α to reduce the translocation of p53 from the nucleus to the cytoplasm [18,31]. In lupulone-treated SW480 cells, p53 was weakly phosphorylated at the level of serine-15 (as compared with SW620 cells) allowing the observed translocation of p53 from the nucleus into the cytoplasm where p53 might exert a protective effect on mitochondrial function [26,27]. This is in accordance with our present data showing that the p53 inhibitor PFT-α by blocking p53 in the nucleus enhanced the apoptotic response induced by lupulone.
Our present report indicates that p53, which carries the same mutations in both cell lines , plays opposing roles in the death signaling pathways triggered by lupulone in SW480 cells and in the derived metastatic SW620 cells (Figure 7). Our data show that in lupulone-treated SW620 cells, p53 is acting in a transcription-dependent manner and plays the role of apoptotic enhancer by favoring the apoptotic response through the activation of the TRAIL-death receptor pathway. Conversely, in lupulone-treated SW480 cells, p53 favors cell survival in an attempt to preserve mitochondrial integrity. However, the prosurvival signals activated by p53 in cells exposed to lupulone is counterbalanced by the previously described  overwhelming apoptotic signaling cascade activated by lupulone. The present data support the view that p53 plays only a marginal role in the regulation of lupulone-triggered apoptosis but demonstrate that the same p53 protein may exert opposite functions (either as an apoptotic or survival factor) in a primary colon adenocarcinoma and in the derived metastatic cells.
The authors thank Behnam Taidi (Carlsberg Breweries A/S, Development Center, Strasbourg, France) for supplying the industrial by-product containing the hop β-acids (lupulone).
1V.L. is supported by a fellowship from the Conseil Régional d'Alsace, France.