Increase in intracellular PGE2 induces apoptosis in Bax-expressing colon cancer cell
© Lalier et al; licensee BioMed Central Ltd. 2011
Received: 28 July 2010
Accepted: 27 April 2011
Published: 27 April 2011
NSAIDs exhibit protective properties towards some cancers, especially colon cancer. Yet, it is not clear how they play their protective role. PGE2 is generally shown as the only target of the NSAIDs anticancerous activity. However, PGE2 known targets become more and more manifold, considering both the molecular pathways involved and the target cells in the tumour. The role of PGE2 in tumour progression thus appears complex and multipurpose.
To gain understanding into the role of PGE2 in colon cancer, we focused on the activity of PGE2 in apoptosis in colon cancer cell lines.
We observed that an increase in intracellular PGE2 induced an apoptotic cell death, which was dependent on the expression of the proapoptotic protein Bax. This increase was induced by increasing PGE2 intracellular concentration, either by PGE2 microinjection or by the pharmacological inhibition of PGE2 exportation and enzymatic degradation.
We present here a new sight onto PGE2 in colon cancer cells opening the way to a new prospective therapeutic strategy in cancer, alternative to NSAIDs.
Prostaglandins are implicated in a wide range of physiological and pathological pathways. Among these pathways, cancer occurrence and development is one of the most debated. It is undoubtable that NSAIDs use was shown to reduce the incidence of some cancers , among which colon cancer took the highest therapeutic advantage . It is unclear, however, how NSAIDs play their protective role. At the tissue level, chronic inflammation is implicated in the development of cancers . Proinflammatory prostaglandins play a role in tumour progression in many ways, namely cell proliferation, survival and migration, immunosuppression and angiogenesis . The anti-inflammatory activity of NSAIDs is thus probably involved in their anti-cancer potency. Yet, at the cellular level, the mechanism by which NSAIDs exert their proapoptotic activity is not clear. PGE2 itself has been shown to play various roles in cell survival and proliferation (reviewed in ). PGE2 induces the activation of several pathways in cancer cells through its interaction with membrane receptors EP(1-4) , and nuclear receptors (PPARδ) , thereby promoting proliferation and survival. Besides, 15-PGDH, the enzyme responsible for its degradation, has been identified as a negative regulator of colon cancer progression . Nevertheless, some models demonstrate a more complex role played by PGE2, since it induces cell death under some circumstances. Thus, it was shown that PGE2 could mediate both neuroprotection and neurotoxicity through the same EP2 receptor, depending on the conditions . Huang and colleagues also demonstrated an EP2/EP4-mediated apoptotic role of PGE2 in fibroblasts . Moreover, PGE2 was also shown to exert opposite effects on colon cancer cells proliferation through different signalling pathways depending on the range of its concentration in the cell culture .
Strikingly, although NSAIDs modulate the production of several prostaglandins, their inhibiting efficiency is classically monitored by the sole measurement of PGE2 secretion. This consideration is very restrictive, since it is known that many processes are regulated by the balance between PGE2 and PGD2, which is also produced downstream of COX-2. Moreover, PGE2 secretion does not strictly reflect PGE2 production since it excludes PGE2 intracellular accumulation and/or degradation. Interestingly, two groups published their results in APC Min/+ mice demonstrating on the one hand that the genetic deletion of mPGES-1, the terminal enzyme responsible for PGE2 synthesis, increased intestinal tumorigenesis , while on the other hand PGE2 treatment induced a raise in intestinal adenoma growth . This apparent discrepancy suggests that PGE2 effects in intestinal tumorigenesis might not be restricted to those observed with extracellular provision.
Besides, we have observed in the glioblastoma  that the overexpression of mPGES-1 was correlated to a longer survival of patients. We have shown in glioblastoma that intracellular PGE2 induced a direct activation of the pro-apoptotic protein Bax, thereby inducing glioblastoma cells apoptosis , whereas extracellular PGE2 did not. The role played by PGE2 in cancer thus appears highly complex, whether in the whole tissue or even in isolated cancer cells. To gain understanding in the signalling of PGE2 in colon cancer cells, we focussed our work on the effect of intracellular PGE2 on the Bax-dependent apoptotic pathway.
Cell culture material was obtained from Gibco (Invitrogen, Cergy Pontoise, France). Unless mentioned, chemical products and reagents were obtained from Sigma (France).
Antibodies were purchased from indicated companies: COX-2 (Cayman, #160107), mPGES-1 (Cayman, #160140), actin (Chemicon, #MAB1501R).
15-PGDH inhibitor (CAY10397) was purchased from Cayman (#70130) (Interchim, France).
3H-PGE2 (0.1 μCi/μl) was purchased from Amersham Biosciences.
Immunoblots were quantified using the ImageJ software (NIH, USA).
Every experiment was repeated at least 3 independent times unless otherwise stated.
Statistical analyses were performed using the GraphPad software (San Diego, CA 92130 USA) (Student unpaired t-test, *: p < 0.05, **: p < 0.01).
Patient materials as well as records (diagnosis, age, sex, date of death) were used with confidentiality according to French laws and recommendations of the French National Committee of Ethic. Tumor samples were collected from adult patients after surgical resection at the Department of anatomo-pathology of the Hospital of Nantes over the years 2002-2003. The clinical information of the patients is summarized in additional file 1 table SI, and additional file 2 table SII. Control tissue was obtained from normal colon tissue found at the periphery of the resected tumor.
Cells were washed twice in PBS, then total RNA was isolated using the RNAwiz (Ambion, Austin, TX, USA) according to the manufacturer's instructions with DNAse I treatment. After RNA quantification using the Nano Drop (Nano Drop ND-1000, Thermo Fisher Scientific, Waltham, MA, USA), the quality of the RNA was determined in an Agilent 2100 Bioanalyzer (Agilent, Palto Alto, CA, USA) using the Labchip RNA 6000 kit. A minimum RNA Integrity Number (RIN) value of 8 was required . Total RNA (1 μg) was reverse transcribed in a final volume of 20 μl using the Superscript II kit (Invitrogen, France). Subsequently the cDNA was diluted to a final concentration of 20 ng/μl, for use in Q-PCR.
The PCR reaction contained 40 ng cDNA in a reaction volume of 25 μl, 1× Brilliant II SYBR Green Q-PCR master mix, 200 nM reverse and forward primers and 30 nM Sybr Green. Thermo-cycling conditions were 95°C for 10 min followed by 40 cycles at 95°C for 1 min, 60°C for 45 s and 72°C for 30 s. Gene expression values were normalized to housekeeping gene (GAPDH) and relative expression values were calculated based on the comparative ΔΔCT-method with adherent cells used as a reference for each cell type.
GAPDH: sense primer: 5'-GAAGGTGAAGGTCGGAGTC-3'
antisense primer: 5'-GAAGATGGTGATGGGATTTC-3'
COX-2: sense primer: 5'-CAGCCATACAGCAAATCC-3'
antisense primer: 5'-ATCCTGTCCGGGTACAAT-3'
mPGES-1: sense primer: 5'-AGGAAGACCAGGAAGTGC-3'
antisense primer: 5'-ACGACATGGAGACCATCTAC-3'
MRP4: sense primer: 5'-AAGTGAACAACCTCCAGTTCCA-3'
antisense primer: 5'-CCGGAGCTTTCAGAATTGAC-3'
15-PGDH: sense primer: 5'-AAGCAAAATGGAGGTGAAGGC-3'
antisense primer: 5'-TGGCATTCAGTCTCACACCAC-3'
Cell culture and transfection
HCT-116 and HCT-116Bax-/- cells (described in ) were grown in McCOY's 5A medium containing 10% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin. SW1116 cells were grown in RPMI medium containing 10% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin. The cells were transfected by a plasmid encoding for the sequence of mPGES-1 cDNA subcloned into pDEST12.2 vector (Invitrogen), or by the mock plasmid containing no coding sequence . Plasmid DNA (5 μg) was introduced into 106 cells by electroporation (GenePulser, Bio-Rad) using 200 V/cm and 250 μF. Transfected cells were selected and further cultured in a medium containing 1 mg/ml G418. The mock-transfected cells were used as a control for the mPGES-1 transfected cells in the expression and viability experiments.
Microinjection was performed as described by Cartron et al. . PGE2 was co-injected with a dextran coupled to a fluorochrome (Oregon Green, Molecular Probes). The instantaneous intracellular concentration of compounds achieved by the microinjection is about one tenth of the initial concentration in the injected solution. The percentage of fluorescent cells exhibiting morphological apoptotic features was evaluated every hour following PGE2 microinjection using an inverted fluorescent microscope (DMIRE2, Leica France).
HCT-116Bax-/- cells were seeded in a 96-well culture plate the day before experiment. 5 μl 3H-PGE2 was added to the cells. Inhibitors of 15-PGDH (CAY10397, 15 μM) and MRP4 (ketoprofen, 1 μM ) were added in every other well. After the indicated incubation time (0 min, 30 min and 1 h), cells were rinsed and harvested. The amount of radiolabeled PGE2 present in the cells was quantified by beta-emission measurement (LS 6500 liquid scintillation counter, Beckman Coulter).
Caspase activation assay
Total cell lysates were carried out with RIPA buffer (PBS, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, proteases inhibitor cocktail (Roche, Meylan, France)) and protein concentrations were measured by the Bradford technique. DEVDase activity was measured using the fluorometric CaspACE Assay System (Promega) and normalized to the sample protein concentration as described previously .
Total cell lysates were obtained with RIPA buffer and separated by SDS-PAGE. Proteins were transferred onto PVDF membranes by semi-dry transfer. Membranes were successively probed with the indicated antibodies and revealed by ECL with peroxidase-coupled secondary antibodies.
SW1116 cells were plated the day before treatment. PGE2 (10 μM) was added to the culture medium. After 10 min, CAY10397 (15 μM) and ketoprofen (1 μM) were added, and cells were treated for 30 h. Cell death was then assessed by trypan blue staining.
Heterogeneity of COX-2 and mPGES-1 expression in human colon cancer
Effect of mPGES-1 overexpression in human colon cancer cell lines
Effect of PGE2microinjection in colon cancer cell
Effect of PGE2intracellular accumulation induced by pharmacological agents.
An extensive amount of data point out that COX-2 and its product PGE2 are actors of cancer promotion and progression. This point has been supported by the fact that the use of COX inhibitors reduces the incidence of several cancers, among which colorectal cancer . Mechanistically, it has been established that PGE2, one of the products of COX-2 activity, could activate several pathways implicated in cancer, namely apoptosis evasion, cell proliferation, migration and angiogenesis. The majority of these effects are mediated through G-coupled EP receptors (EP1-4) . Nevertheless, conflicting results tend to demonstrate that PGE2 is much more versatile than what was initially thought.
Besides, caution should be used when considering COX inhibitors as "anti-PGE2" compounds. The inhibition of COX-2, even with the use of selective COX-2 inhibitors, definitely has larger consequences than a decrease in PGE2 synthesis since several prostaglandins arise from COX-2 activity. For instance, Thoren and Jakobsson  demonstrated that COX-2 inhibitors had a various ability to inhibit mPGES-1 activity. As a consequence, COX-2 inhibitors not only modulate COX-2 activity but also the potential coupling of COX-2 and mPGES-1 activity; they consequently not only modulate PGH2 production but also the ratio between the PGH2 products, among which PGE2 and PGD2, which are known to exert opposite effects on Bax activation .
Given the multiplicity of the physiological functions of prostaglandins and the very subtle regulatory processes which can hardly be predicted in the whole, we need a deeper understanding of the pathways in which the products of COX are implicated in cell signalling, in the tissue and in the body, before safely using NSAIDs as anti-cancer therapeutic adjuvants. More disturbing is the observation that the anti-proliferative effects of COX-2 inhibitors on cancer cells have also been demonstrated in COX-2-deficient cells [18–21], suggesting that the role of COX-2 and its product PGE2 in cancer might have been overvalued based on the effects of pharmacological COX-2 inhibitors.
We show here that the overexpression of mPGES-1, the enzyme responsible for PGE2 synthesis downstream of COX-2, sensitizes isolated colon cancer cells to apoptosis in vitro. We also demonstrate that cell death can be induced in colon cancer cells by increasing the intracellular content in PGE2, either through direct microinjection or through the inhibition of PGE2 intracellular exportation and degradation, provided the cells express the protein Bax. Indeed, taking into account the data of Reid and colleagues , which showed that several NSAIDs exert an inhibitory activity on MRP4 at concentrations inferior to those used for COX inhibition, we showed that ketoprofen could induce a PGE2-dependent cell death, even if MRP4 inhibition might inhibit the efflux of other compounds from the cells; this could partly explain the disastrous cardiac side effects observed during long-term NSAID treatments. Similarly, we did not explore the consequence of 15-PGDH inhibition on the concentration of other prostaglandins, but we showed that the cell death was considerably increased by the adding of PGE2 in the cell culture, demonstrating that PGE2 was at least partly responsible for the apoptosis induced. These results are consistent with what we have previously described in glioblastoma . To our comprehension, our results also bring a possible explanation to some of the conflicting results observed between extracellular PGE2 treatments and modulations of PGE2 production (see  and  for example). With the care to be as representative as possible for colon cancers, our in vitro work was realised with four colon cancer cell lines, two of which presented LOH (SW1116 and HT29) whereas the other two were classified MSI-positive (HCT-116 and HCT-8)[22, 23].
What could be the rationale of these ambiguous properties exhibited by PGE2? An attractive concept was recently described by Li and colleagues . They report that executive caspases, key players of apoptotic cell death, are necessary for wound healing. The activation of these caspases in injured cells is responsible for PGE2 synthesis and exportation. When released at the wounded site, PGE2 stimulates stem cells proliferation and tissue regeneration. PGE2 might thus be regarded as a danger signal emerging from dying cells. Our understanding of the mechanism is that newly produced, intracellular PGE2 is able to sensitize the cells to death through the activation of the apoptotic protein Bax. In the cells where the death signals overwhelm the resistance capacities, caspases are activated and apoptosis occurs. Meanwhile, PGE2 production and release in the environment is increased; PGE2 thus exerts its antiapoptotic, proliferative and migratory role on the neighbouring cells through the EP receptors pathway. In the context of a tumour, the surviving cells become more resistant to a subsequent insult. PGE2 activity in tumour cells would thus follow a two-step mechanism: first, intracellular PGE2 participates in apoptotic cell death; second, secreted PGE2 has an autocrine or paracrine protective and stimulating activity, respectively on the producing cell if the integration of death signals is compatible with survival, or on the neighbouring cells if cell death is induced, with an amplification loop in PGE2 production through executive caspases activity. The important consequence of this mechanism is that PGE2 exportation from cancer cells is the most detrimental determinant in the role played by PGE2 in tumour progression. An alternative to COX-2 inhibitors as adjuvant anti-cancer therapies might thus be the use of drugs inhibiting PGE2 efflux from the cancer cells. The potential of MRP4 inhibitors in enhancing classical therapies would thereby be worth questioning.
Our present work demonstrates that intracellular PGE2 can exert a pro-apoptotic, Bax-dependent apoptosis in colon cancer cell lines in vitro. We thereby bring an additional level of complexity in the highly complex role played by PGE2 in colon cancer progression. We also suggest that MRP4 inhibition might be a valuable adjuvant strategy to colon cancers treatments.
non steroidal anti-inflammatory drugs
- PGE2 :
- PGD2 :
microsomal prostaglandin E2 synthase-1
We are grateful to Anne Jarry for the gift of colon tissue samples and patients information. We thank Dr Jacques Le Pendu for the generous gift of HT29 and HCT-8 cell lines.
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