Pancreatic cancer remains as one of the most deadly cancers, and responds poorly to current therapies. The prognosis is extremely poor, with a 5-year survival of less than 5%. Therefore, search for new effective therapeutic drugs is of pivotal need and urgency to improve treatment of this incurable malignancy. Synthetic alkyl-lysophospholipid analogs (ALPs) constitute a heterogeneous group of unnatural lipids that promote apoptosis in a wide variety of tumor cells. In this study, we found that the anticancer drug edelfosine was the most potent ALP in killing human pancreatic cancer cells, targeting endoplasmic reticulum (ER). Edelfosine was taken up in significant amounts by pancreatic cancer cells and induced caspase- and mitochondrial-mediated apoptosis. Pancreatic cancer cells show a prominent ER and edelfosine accumulated in this subcellular structure, inducing a potent ER stress response, with caspase-4, BAP31 and c-Jun NH2-terminal kinase (JNK) activation, CHOP/GADD153 upregulation and phosphorylation of eukaryotic translation initiation factor 2 α-subunit that eventually led to cell death. Oral administration of edelfosine in xenograft mouse models of pancreatic cancer induced a significant regression in tumor growth and an increase in apoptotic index, as assessed by TUNEL assay and caspase-3 activation in the tumor sections. The ER stress-associated marker CHOP/GADD153 was visualized in the pancreatic tumor isolated from edelfosine-treated mice, indicating a strong in vivo ER stress response. These results suggest that edelfosine exerts its pro-apoptotic action in pancreatic cancer cells, both in vitro and in vivo, through its accumulation in the ER, which leads to ER stress and apoptosis. Thus, we propose that the ER could be a key target in pancreatic cancer, and edelfosine may constitute a prototype for the development of a new class of antitumor drugs targeting the ER.
Pancreatic adenocarcinoma is one of the most aggressive cancers. Pancreatic cancer is often detected at an advanced stage and prognosis is extremely poor, with a median survival of 4–6 months (Li et al., 2004). It represents ∼10% of all gastrointestinal malignancies (Neoptolemos et al., 2003), and it is the fourth most common cause of death in Western countries (Jemal et al., 2007). Most patients with diagnosed pancreatic cancer do not benefit from surgery and frequently need palliative chemotherapy (van Riel et al., 1999). Standard treatments for advanced disease include 5-fluorouracil and gemcitabine. However, even gemcitabine, considered to be the gold standard for pancreas cancer, has a response rate of less than 20% (Li et al., 2004).
The endoplasmic reticulum (ER) is an organelle responsible for several important cellular functions, including protein and lipid biosynthesis, post-translational modification, folding and assembly of newly synthesized secretory proteins and cellular calcium store. Various conditions can disturb ER functions, leading to a series of events collectively termed as ER stress. An excessive ER stress leads to an accumulation of misfolded proteins in the ER lumen, which initiates the unfolded protein response (UPR) to restore normal ER function. However, persistent ER stress can switch the cytoprotective functions of UPR into cell death-promoting mechanisms, leading to the triggering of ER-dependent apoptotic cascades. One of the features of pancreatic cells is a highly developed ER, apparently due to a heavy engagement in insulin secretion (Oyadomari et al., 2002). ER stress is directly related to pancreatic cell dysfunction and death by apoptosis, during the progression of type 1 and type 2 diabetes mellitus and Wolfram syndrome (Fonseca et al., 2009). Nevertheless, ER stress sensors do not directly cause cell death, but rather initiate the activation of downstream molecules, such as CHOP (C/EBP homologus protein)/growth arrest and DNA damage-inducible gene 153 (GADD153) or c-Jun NH2-terminal kinase (JNK), which further push the cell down the path of death. Three main pathways of ER stress-induced apoptosis are known, namely: (1) upregulation of the transcription factor CHOP/GADD153 (Ron and Habener, 1992); (2) JNK activation (Urano et al., 2000); (3) activation of caspase-12 in murine systems or caspase-4 in human cells (Hitomi et al., 2004). These three pathways all end in caspase cascade activation followed by the induction of apoptosis.
Accumulation of unfolded proteins, leading to ER stress, can be induced by anticancer chemotherapeutic agents such as bortezomib (Fribley et al., 2004), geldanamycin (Mimnaugh et al., 2004), cisplatin (Mandic et al., 2003) and cannabinoids (Carracedo et al., 2006). Also, chronic exposure to long-chain free fatty acids induces ER stress and cell death in pancreatic beta-cells (Cunha et al., 2008). Synthetic alkyl-lysophospholipid analogs (ALPs) are a novel class of unnatural lipids with promising anticancer activity, including clinically relevant drugs such as miltefosine, perifosine and erucylphosphocholine, which act at the cell membrane level (Gajate and Mollinedo, 2002; Mollinedo et al., 2004). Edelfosine (1-O-octadecyl-2-O-methoxy-rac-glycero-3-phosphocholine), considered to be the ALP prototype compound, has been shown to induce apoptosis in malignant cells in a rather selective way through the involvement of lipid rafts and ER (Mollinedo et al., 1997; Gajate et al., 2000b, 2004, 2009a; Gajate and Mollinedo, 2001, 2007; Mollinedo and Gajate, 2006, 2010; Nieto-Miguel et al., 2007).
Because the pancreatic cancer cells show prominent ER (Klimstra et al., 1992; Skarda et al., 1994), we wondered whether edelfosine might be active against pancreatic cancer by targeting ER. Hence, we investigated the in vitro and in vivo action of edelfosine on pancreatic cancer as well as its mechanism of action. In this work, using different in vitro and in vivo experimental approaches, we show that edelfosine behaves as an effective anticancer agent that induces apoptotic cell death of pancreatic cancer cells, via accumulation of the drug at the ER, leading to ER stress response and eventually to apoptosis.
Induction of apoptosis by edelfosine and other ALPs in human pancreatic cancer cells
We have recently found that the pharmacologically relevant concentration in the plasma of edelfosine ranges between 10 and 20 μM (Estella-Hermoso de Mendoza et al., 2009; Mollinedo et al., 2010). Thus, we first analyzed the pro-apoptotic activity of edelfosine and different ALPs, including perifosine, miltefosine and erucylphosphocholine at 10 and 20 μM, on a number of human pancreatic cancer cells. Our data indicated that edelfosine induced cell death in a dose- and time-dependent manner (Figure 1a). We found that edelfosine was the only ALP that promoted apoptosis at 10 μM in all the pancreatic cancer cells (Figure 1a), whereas the other ALPs did not induce cell killing at this drug concentration, even after 72 h of incubation (data not shown). At 20 μM, we found that ALPs ranked edelfosine>perifosine≫erucylphosphocholine⩾miltefosine in their capacity to induce apoptosis in the pancreatic BxPC-3, Capan-2, CFPAC-1 and HuP-T4 cancer cells (Figure 1b). We included in our analysis the structurally related inactive edelfosine analog 1-O-octadecyl-rac-glycero-3-phosphocholine (ET-18-OH) (Mollinedo et al., 1997; Gajate et al., 1998), in which the methoxy group in the sn-2 position was replaced by an OH group. We found that, unlike edelfosine and perifosine, the other ALPs rendered low figures of apoptosis similar to ET-18-OH (Figure 1b). Thus, edelfosine and perifosine were the only ALPs with pro-apoptotic activity against pancreatic cancer cells.
Edelfosine accumulates in the ER of pancreatic cancer cells
Because edelfosine behaved as the most potent ALP in inducing cell death in the pancreatic cancer cells, we next analyzed its mechanism of action. First, we found that all pancreatic cancer cell lines used in this study incorporated high amounts of drug (Figure 1c). Next, we found that the new fluorescent edelfosine analog 1-O-[11′-(6″-ethyl-1″,3″,5″,7″-tetramethyl-4″,4″-difluoro-4″-bora-3a″,4a″-diaza-s-indacen-2″-yl)undecyl]-2-O-methyl-rac-glycero-3-phosphocholine (Mollinedo et al., 2011) accumulated in the ER of HuP-T4 and Capan-2 cells (Figure 2a), as assessed using a version of red fluorescence protein targeted to the ER lumen (ER-targeted red fluorescence protein), which completely co-localized with the ER marker calreticulin (Klee and Pimentel-Muinos, 2005). Incorporation of fluorescent 1-O-[11′-(6″-ethyl-1″,3″,5″,7″-tetramethyl-4″,4″-difluoro-4″-bora-3a″,4a″-diaza-s-indacen-2″-yl)undecyl]-2-O-methyl-rac-glycero-3-phosphocholine into the cells was blocked by adding the parent drug edelfosine (data not shown), thus behaving as a reliable edelfosine fluorescent analog to visualize the subcellular location of the drug ‘in situ’.
Edelfosine induces ER stress response in pancreatic cancer cells
We next analyzed whether this drug induced an ER stress response leading to apoptosis in the HuP-T4 cells. To this aim, we examined the effect of edelfosine on a number of ER stress-associated markers, including caspase-4, CHOP/GADD153, BAP31, GRP78/BiP and eukaryotic translation initiation factor 2 α-subunit (eIF2α). Human caspase-4 is a potential homolog of murine caspase-12 involved in ER stress (Hitomi et al., 2004). As shown in Figure 2b, edelfosine treatment induced caspase-4 activation, as assessed by cleavage of procaspase-4 into the 20-kDa active form. CHOP/GADD153 activates transcription of several genes that potentiate apoptosis during ER stress (Oyadomari and Mori, 2004). CHOP/GADD153 expression, together with phosphorylation of eIF2α, were strongly induced in the HuP-T4 cells following edelfosine treatment (Figure 2b). BAP31 is an integral membrane protein of the ER that regulates ER-mediated apoptosis through its caspase-8-mediated cleavage into a 20-kDa fragment, which directs pro-apoptotic signals between the ER and mitochondria (Breckenridge et al., 2003). Here, we found that BAP31 was cleaved into the p20 fragment upon edelfosine treatment (Figure 2b). Edelfosine did not upregulate Grp78/BiP (Figure 2b), a major ER chaperone that binds Ca2+ and promotes tumor proliferation, survival, metastasis and resistance to a wide variety of therapies (Li and Lee, 2006). ER stress has been shown to induce activation of Bax (Zong et al., 2003), and we found Bax activation in response to edelfosine treatment in pancreatic cancer cells, by using an anti-Bax monoclonal antibody that recognized the active form of Bax (Figure 2b). Previous studies have shown that ASK1-mediated JNK activation is crucial for ER-induced apoptosis (Nishitoh et al., 2002). We found here that edelfosine induced a potent and sustained activation of JNK in all pancreatic cancer cell lines (Figure 2c). Pre-incubation with the caspase-4 inhibitor z-LEVD-fmk, or the specific JNK inhibitor SP600125, diminished edelfosine-induced apoptosis (Figures 2d and e), when used at concentrations that prevented edelfosine-induced caspase-4 and JNK activation (data not shown). These data reveal that edelfosine accumulates in the ER of pancreatic cancer cells, and induces an ER stress response.
Edelfosine induces sXBP1 expression
When the UPR is induced during ER stress, the ER-resident transmembrane kinase-endoribonuclease inositol-requiring enzyme 1 (IRE1) is activated, leading to site-specific splicing to form spliced XBP1 mRNA (sXBP1), by removing a 26-nucleotide internal sequence from unspliced XBP1 (uXBP1) mRNA. The presence in this 26-nucleotide fragment of a PstI restriction site further allowed us to distinguish between both XBP1 forms by restriction analysis of PCR-amplified complementary DNA, and thus to assay for ER stress response activation (Hirota et al., 2006). By reverse transcriptase–PCR we found the induction of sXBP1 mRNA following treatment of BxPC-3 cells with 20 μM edelfosine (Figure 2f). Similar data were obtained by PstI restriction analysis (data not shown). Because sXBP1 is a key modulator of the UPR, our results suggest that edelfosine induces ER stress and UPR signaling in pancreatic cancer cells.
UPR is known as a pro-survival response to reduce the accumulation of unfolded proteins and restore normal ER function. However, when persistent, ER stress can switch the cytoprotective functions of UPR into cell death-promoting mechanisms. Pre-incubation of BxPC-3 cells for 1 h with different concentrations (up to 2 mM) of dithiothreitol (DTT), a widely used ER and UPR stress inducer, before edelfosine addition, did not cause apoptosis (Supplementary Figure S1a). Pre-treatment of BxPC-3 cells with DTT for 1 h, followed by washing off DTT and subsequent incubation with edelfosine for 24 h, slightly reduced the percentage of apoptotic cells as compared with cells treated only with edelfosine without DTT pre-treatment (Supplementary Figure S1a). However, this reduction was not statistically significant (P=0.15).
By using the primers indicated in Supplementary data, the sXBP1 form generates a 414-bp fragment, while the uXBP1 form generates a 440-bp fragment, which contains a PstI restriction site leading to the generation of two bands of 294 and 146 bp following PstI digestion. However, sXBP1, lacking the restriction site is resistant to PstI digestion and hence only one band of 414 bp is obtained. As shown in Supplementary Figure S1b, sXBP1 was readily observed in pancreatic cancer cells incubated with DTT, edelfosine or DTT+edelfosine. Edelfosine by itself was a potent inducer of sXBP1 (Supplementary Figure S1b). These results suggest that edelfosine activates UPR by activating the IRE1–XBP1 branch of the UPR pathway. Taken together, our data might suggest that previous induction of UPR does not inhibit edelfosine-induced apoptosis. Thus, our data suggest that edelfosine action on ER is persistent and leads to the triggering of apoptotic signals that may override protective UPR mechanisms.
Edelfosine induces changes in ER calcium level
Because ER has a critical role in controlling cellular Ca2+ levels, we next analyzed how edelfosine affected [Ca2+]ER. Reconstitution of ER-targeted aequorin with a semisynthetic prosthetic group, coelenterazine n, required previous depletion of Ca2+ of the ER to prevent aequorin consumption during the reconstitution process (Montero et al., 1997b). Once aequorin was reconstituted, the ER was refilled again with Ca2+ by perfusing the cells with extracellular medium containing 1 mM Ca2+. This led to an increase in the [Ca2+]ER, that reached a steady state of around 250 μM within 3–4 min in the BxPC-3 cells (Figure 3a). Addition of edelfosine induced a large increase in [Ca2+]ER, which nearly doubled the previous values within 10–15 min (Figure 3a). This effect was time dependent. Adding edelfosine 15 min before starting ER refilling induced a huge [Ca2+]ER increase reaching levels above 900 μM (Figure 3b). However, longer incubation times (30 and 60 min) with edelfosine induced only a twofold increase of the steady-state ER calcium levels (Figure 3b). These results suggest a progressive decrease in the ability of the ER to take up Ca2+, and therefore these data could indicate that edelfosine affects one of the major functions of the ER, by altering the ER calcium level in a time-dependent way, which could have consequences in the cell's fate. Thus, edelfosine affects a number of ER-related processes and functions.
Involvement of caspases 8 and 10, and cleavage of Bid in edelfosine-induced apoptosis
BAP31 cleavage by edelfosine (Figure 2b) suggested the involvement of caspase-8 in the process. Western blot analyses showed early cleavage of pro-caspases 8 and 10 in pancreatic cancer cells after edelfosine treatment, as assessed by the appearance of p18-active caspase-8 and p23-active caspase-10 forms (Figure 4a). Pre-incubation of cells with caspase-8 inhibitor z-IETD-fmk and caspase-10 inhibitor z-AEVD-fmk blocked edelfosine-induced apoptosis (Figure 4b). Thus, both caspases 8 and 10 are involved in the apoptosis response induced by edelfosine in pancreatic cancer cells.
Bid is a potent pro-apoptotic Bcl-2 family member which, upon proteolytic activation by caspases 8 or 10, translocates onto mitochondria to promote activation of the Bax/Bak subgroup of the apoptotic Bcl-2 family proteins, and thereby contributes to the release of cytochrome c (Kuwana et al., 2005). We found Bid cleavage after the edelfosine treatment, by using an anti-Bid antibody that recognized both full-length and 18-kDa cleaved forms (Figure 4c). These results suggested that Bid might have a role in edelfosine-induced apoptosis by transferring signals to the mitochondria.
Involvement of mitochondria in edelfosine-induced apoptosis in human pancreatic cancer cells
Because the above results showed that edelfosine promoted the cleavage of BAP31 and Bid into p20 and p18 fragments respectively, acting as signal transmitters toward mitochondria in apoptosis signaling, we next analyzed the role of mitochondria in the apoptotic response triggered by edelfosine in pancreatic cancer cells. Once cytochrome c is released from mitochondria to the cytosol during apoptosis, it leads to the formation of the apoptotic protease-activating factor 1 (Apaf-1)/caspase-9 complex, initiating an apoptotic protease cascade that promotes degradation of key structural proteins, including poly(ADP-ribose) polymerase, and eventually causing DNA fragmentation and apoptosis. We found that edelfosine induced caspase-9, -7 and -3 activation, assessed by cleavage of pro-caspase-9, -7 and -3 into their respective active forms, as well as proteolysis of the caspase-3 and caspase-7 substrate poly(ADP-ribose) polymerase in all the pancreatic cancer cells used in this study (Figure 5a). The caspase-9 inhibitor z-LEHD-fmk, and the caspase-3 inhibitor Ac-DEVD-CHO, inhibited the apoptotic death of pancreatic cancer cells induced by edelfosine (Figure 5b). In addition, inhibition of caspases by the broad caspase inhibitor z-VAD-fmk completely abrogated edelfosine-induced apoptosis (Figure 5b). These findings indicate that edelfosine-induced apoptosis in human pancreatic cancer cells is caspase dependent.
As shown in Figure 5c, edelfosine treatment induced a marked release of cytochrome c from the mitochondria to the cytosol in the BxPC-3 pancreatic cancer cells. Similarly, we also found mitochondrial cytochrome c release in Capan-2, CFPAC-1 and HuP-T4 pancreatic cancer cells following edelfosine treatment (Supplementary Figure S2). Because Bcl-XL acts as a safeguard of mitochondria, preventing cytochrome c release and apoptosis, we stably transfected the BxPC-3 cells with pSFFV-bcl-xL plasmid (BxPC-3-Bcl-XL), containing the human bcl-xL open-reading frame, or with control pSFFV-Neo plasmid (BxPC-3-Neo). The BxPC-3-Neo cells behaved similarly to the non-transfected BxPC-3 cells regarding all parameters studied. The BxPC-3-Neo cells expressed a small level of endogenous Bcl-XL, whereas a high expression of this protein was observed in BxPC-3-Bcl-XL cells (Figure 5d). Whereas the BxPC-3-Neo cells underwent apoptosis after treatment with edelfosine, Bcl-XL overexpression prevented edelfosine-induced apoptosis (Figure 5e). Edelfosine induced mitochondrial release of cytochrome c in BxPC-3-Neo cells (Figure 5f), but cytochrome c release was highly diminished in BxPC-3-Bcl-XL cells (Figure 5f). These data show that mitochondria are involved in the edelfosine-induced pancreatic cancer cell death.
In vivo antitumor effect of edelfosine in pancreatic cancer xenograft models
We next evaluated the effect of orally administered edelfosine in two pancreatic cancer xenograft animal models. Following toxicity analyses with CB17-severe combined immunodeficient and BALB/c mice (data not shown), we found that a daily oral administration dose of 30 mg/kg edelfosine was well tolerated, 45 mg/kg being the maximum tolerated dose. CB17-severe combined immunodeficient mice were inoculated with 5 × 106 Capan-2 or HuP-T4 cells. When tumors were palpable in the two pancreatic cancer animal models, mice were randomly assigned to cohorts of eight mice each, receiving a daily oral administration of edelfosine (30 mg/kg) or an equal volume of vehicle (water). Serial caliper measurements were done every 3 days to calculate tumor volume until mice were killed (Figure 6a). A comparison of tumors isolated from untreated control and drug-treated Capan-2 or HuP-T4-bearing mice, at the end of the treatment, rendered a remarkable anti-pancreatic cancer activity of edelfosine (Figures 6b and c), with a statistically significant (P<0.05) reduction of ∼57 and ∼66% in the tumor weight in both Capan-2 (233±50 vs 539±106 mg, for edelfosine-treated vs untreated mice, n=8) and HuP-T4 (305±67 vs 896±97 mg, for edelfosine-treated vs untreated mice, n=8) animal models (Figure 6b). Likewise, a statistically significant (P<0.05) reduction of ∼60 and ∼72% in tumor volume was also detected in both Capan-2 (240±48 vs 600±124 mm3, for edelfosine-treated vs untreated mice, n=8) and HuP-T4 (312±68 vs 1114±145 mm3, for edelfosine-treated vs untreated mice, n=8) animal models (Figure 6b). Interestingly, while tumors from drug-free mice showed a highly vascular appearance, tumors from edelfosine-treated mice were pale and poorly vascularized (Figure 6c). Organ examination at necropsy did not reveal any apparent toxicity (data not shown), and no significant differences in mean body weight were detected between drug-treated and drug-free control animals (2–3% of body weight loss in the drug-treated vs drug-free control groups).
In vivo identification of apoptosis and ER stress in pancreatic tumors following edelfosine oral treatment
Histological patterns of tumors isolated from control drug-free tumor-bearing animals revealed a relative uniformity in cell size and morphology (Figure 7a). In contrast, examination of hematoxylin and eosin-stained sections of tumors from mice orally treated with edelfosine showed the presence of irregular, large and medium-sized giant cells with eosinophilic cytoplasm. The nuclei in dying cells were either pyknotic or displayed nuclear fragmentation characteristic of cell death by apoptosis (Figure 7a). Induction of apoptosis was further supported by performing TUNEL apoptosis assay in the tumor sections. No TUNEL fluorescence was detected in the tumor xenografts of the control drug-free group (Figure 7b). However, a strong TUNEL-positive signal, indicating apoptotic cells, was detected in the edelfosine-treated group (Figure 7b). Our data showed a significant difference in the percentage of TUNEL-positive cells between the control drug-free and drug-treated groups, namely 2.2±0.6 vs 35.0±5.4%.
The anti-activated caspase-3-specific antibody selectively labeled the cytoplasm of cells that had a morphology consistent with apoptosis in tumors from drug-treated mice (Figure 7c), while lack of staining was observed in the control drug-free group. Our data showed that the activated caspase-3 labeling index in the tumor tissue of the drug-treated mice was significantly higher (40±5.6%) than that in the drug-free mice (1.2±0.1%) (Figure 7c).
Interestingly, the ER stress-associated marker CHOP/GADD153 was visualized by immunohistochemistry in the nuclei of cells in the pancreatic tumors isolated from edelfosine-treated mice, indicating a strong ER stress response in the tumor sections. However, CHOP/GADD153 staining was absent in tumors derived from control drug-free mice (Figure 7d).
Pancreatic adenocarcinoma responds poorly to current therapies and remains as an incurable malignancy. This makes this tumor especially challenging for searching novel effective anticancer drugs. Our in vitro and in vivo data indicate that edelfosine is a potent antitumor agent against pancreatic tumor cells, and highlight the importance of ER as a target for the treatment of pancreatic cancer. We have found that edelfosine behaves as the most potent ALP in killing human pancreatic cancer cells by targeting ER. Perifosine was also active against pancreatic cancer cells, but other ALPs, such as miltefosine and erucylphosphocholine, as well as the structurally related inactive molecule ET-18-OH, failed to induce cell death in these cells, underlining the importance of the molecular structure of edelfosine for its anti-pancreatic cancer activity. The studies reported here show for the first time in vitro and in vivo evidences for the induction of ER stress in the mechanism of action of an ER-targeted antitumor drug in pancreatic cancer, suggesting that this could be a promising therapeutic approach in the treatment of pancreatic cancer.
We have found evidence that edelfosine was working in vivo in a similar manner to that observed in vitro. Oral administration of edelfosine showed a remarkable in vivo antitumor and pro-apoptotic activity, promoting a potent apoptosis response in human tumor xenografts in severe combined immunodeficient mice, as assessed by morphological changes, TUNEL assay and caspase-3 activation. Our data represent the first demonstration of the in vivo pro-apoptotic activity of edelfosine, which further supports the notion that the antitumor action of this ALP highly depends on its ability to promote apoptosis in tumors. Edelfosine treatment led to dramatic tumor regression in pancreatic tumor animal models, and tumors became smaller and poorly vascularized, which could be in agreement with reports showing an antiangiogenic effect of edelfosine (Zerp et al., 2008).
Pancreatic cancer cells have been reported to show prominent ER (Klimstra et al., 1992; Skarda et al., 1994). Here, we report for the first time that edelfosine accumulates in the ER of human pancreatic cancer cells and triggers a prolonged ER stress response, leading to apoptosis. Unlike several anticancer drugs that induce ER stress in an indirect way, here we found that edelfosine accumulates in the ER of human pancreatic cancer cells and promotes a number of changes in ER-regulated homeostatic processes, leading to the triggering of sundry ER-derived apoptotic events that eventually converge on the mitochondria. Edelfosine also upregulates sXbp1, a key modulator of the UPR, but activation of this protective response during ER stress seems not to be enough to prevent apoptosis. The role of UPR activation in edelfosine-induced apoptosis remains to be elucidated, and we cannot rule out that upregulation of the above UPR marker might merely be a correlative phenomenon. The results obtained measuring the effects of edelfosine on [Ca2+]ER in the BxPC-3 cells suggest that edelfosine induces a significant deregulation of global Ca2+ homeostasis. We have previously shown that edelfosine increases cytosolic [Ca2+] in HeLa cells (Nieto-Miguel et al., 2007). On these grounds, and taking into account the interaction of edelfosine with cell membranes and lipids (Gajate et al., 2004, 2009a, 2009b; Zaremberg et al., 2005; Mollinedo and Gajate, 2006; Torrecillas et al., 2006; Busto et al., 2007, 2008; Ausili et al., 2008; Mollinedo et al., 2010), it might be envisaged that the increase in cytosolic [Ca2+] could be due to an increased permeability of the plasma membrane, resulting in Ca2+ entry from the extracellular medium, which it then leads to the herein reported increase in [Ca2+]ER, well above the steady state levels in control cells. The effect on [Ca2+]ER may seem puzzling, because ER stress has been rather associated with ER-stored Ca2+ release. However, our present results also indicate that this Ca2+ pumping into the ER, following edelfosine incubation, is progressively decreased with time. These results might reflect an altered function of the ER, leading to a gradual impairment in its capacity to take up Ca2+ and hence to Ca2+ homeostasis deregulation in the ER. All these aspects deserve further studies on the role of Ca2+ in the pro-apoptotic action of edelfosine on pancreatic cancer cells.
Figure 8 depicts a model for the involvement of ER in edelfosine-induced apoptosis in pancreatic cancer cells. Our previous (Nieto-Miguel et al., 2007) and present data suggest that edelfosine causes a gradual alteration in calcium homeostasis. Transient increase in cytosolic Ca2+ stimulates Ca2+ uptake by mitochondria (Rizzuto and Pozzan, 2006), which contributes to mitochondrial membrane permeability transition, releasing apoptogenic proteins. Here, we have found that edefosine induces cytochrome c release from mitochondria in pancreatic cancer cells. Cytochrome c released from mitochondria binds to Apaf-1 and pro-caspase-9 to form the so-called apoptosome (Riedl and Salvesen, 2007). This complex catalyzes the activation of caspases that executes the apoptotic cell death program. The herein reported abrogation of edelfosine-induced apoptosis, and mitochondrial cytochrome c release, by Bcl-XL overexpression indicates an essential role for mitochondria in the induction of apoptosis by this anticancer drug in pancreatic cancer cells. ER stress-induced processing of procaspase-9 can also occur in the absence of cytochrome c release and in Apaf-1-null fibroblasts (Rao et al., 2002), suggesting that caspase-4 can directly trigger caspase-9 activation and apoptosis independent of the mitochondrial cytochrome c/Apaf-1 pathway, at least in certain cell types. Our results suggest that both caspase-4- and apoptosome-mediated signaling pathways are involved in edelfosine-induced apoptosis (Figure 8). A prolonged ER stress during exposure to edelfosine leads to the induction of the pro-apoptotic transcription factor CHOP/GADD153. Here, we found both in vitro and in vivo evidence for the upregulation of CHOP/GADD153 following edelfosine treatment in pancreatic cancer cells, whereas the level of GRP78/BiP protein was not significantly altered, tipping the balance in favor of an ER stress-induced cell death. Thus, these results imply that induction of CHOP/GADD153 expression is closely associated with the progression of apoptosis during exposure of pancreatic cancer cells to edelfosine. Our data suggest that edelfosine induces the cleavage of BAP31 (an integral membrane protein of the ER) in pancreatic cancer cells, with the formation of p20 fragment that directs pro-apoptotic signals between ER and mitochondria, resulting in the discharge of Ca2+ from the ER and its concomitant uptake into the mitochondria. Also, edelfosine promotes phosphorylation of eIF2α, another typical response to ER stress. In addition, we found that ER stress-associated caspase-4 was activated before the onset of apoptosis following edelfosine treatment in pancreatic cancer cells. Caspase-4 inhibition abrogated edelfosine-induced apoptosis, suggesting that caspase-4 is required for the triggering of cell death. In addition, edelfosine-induced apoptosis in pancreatic cancer cells involves caspase-8 activation and persistent activation of JNK.
Members of the Bcl-2 family are also involved in the regulation of cell death induced by ER stress (Oakes et al., 2006). In normal conditions, mammalian cells express low levels of Bax, which is predominantly a soluble monomeric protein in the cytosol (Hsu et al., 1997). Under ER stress conditions, a significant fraction of Bax may translocate from cytosol to membrane fractions, in particular the mitochondrial membrane (Hsu et al., 1997). Insertion of Bax into mitochondria causes the release of cytochrome c and Ca2+ to the cytosol. Our previous (Mollinedo et al., 1997; Nieto-Miguel et al., 2007) and present data indicate that edelfosine does not modify the expression of antiapoptotic Bcl-2 and Bcl-XL genes, whereas Bax is activated. In addition, cells lacking both Bax and Bak have been shown to be resistant to ER stress-induced apoptosis (Zong et al., 2001). We have previously reported that bax−/−/bak−/− double-knockout cells fail to undergo edelfosine-induced ER-stored Ca2+ release and apoptosis (Nieto-Miguel et al., 2007). Taken together, our previous and present results suggest a role for Bax in edelfosine-induced apoptosis in pancreatic cancer cells.
Overall, the results reported here indicate that edelfosine exerts its pro-apoptotic action in pancreatic cancer cells, both in vitro and in vivo, through its accumulation in the ER that leads to a sustained ER stress and eventually to cell death. These data suggest that ER targeting by edelfosine may represent a promising new framework in the treatment of currently incurable pancreatic cancer. Our results also provide a rationale for searching new effective agents targeting ER to treat pancreatic cancer.
Materials and methods
Detailed information on the reagents used is included in Supplementary data.
Detailed information on the cell lines used is included in Supplementary data.
BxPC-3 cells (1–2 × 105) were transfected with 8 μg of pSFFV-Neo or pSFFV-bcl-xL expression vector as previously described (Mollinedo et al., 1997), using Lipofectin reagent (Life Technologies, Carlsbad, CA, USA). Transfected clones were selected by growth in the presence of 600 μg/ml of G418 (Sigma, St Louis, MO, USA), then cultured at 250 μg/ml of G418 and monitored by western blotting using the 2H12 anti-29 kDa Bcl-XL monoclonal antibody (BD Biosciences Pharmingen, San Diego, CA, USA).
Drug uptake was measured as previously described (Mollinedo et al., 1997) with slight modifications. After incubating 106 cells/ml with 10 μM edelfosine plus 0.05 μCi/ml [3H]edelfosine for 1 h, and subsequent exhaustive washing (five times) with 1% bovine serum albumin–phosphate-buffered saline, 0.1 ml of 0.2% Triton X-100 was added to the cell pellets, and the incorporated radioactivity was counted in a beta-counter by mixing with water-miscible liquid scintillation cocktail. [3H]edelfosine (specific activity, 42 Ci/mmol) was synthesized by tritiation of the 9-octadecenyl derivative (Amersham Buchler, Braunschweig, Germany).
Drug subcellular localization
The subcellular localization of edelfosine in pancreatic cancer cells was examined with the newly synthesized edelfosine fluorescent analog 1-O-[11′-(6″-ethyl-1″,3″,5″,7″-tetramethyl-4″,4″-difluoro-4″-bora-3a″,4a″-diaza-s-indacen-2″-yl)undecyl]-2-O-methyl-rac-glycero-3-phosphocholine (Mollinedo et al., 2011), a kind gift from F. Amat-Guerri and A. U. Acuña (Consejo Superior de Investigaciones Científicas, Madrid, Spain). ER was visualized by transfecting cells with 4 μg of a plasmid encoding ER-targeted red fluorescence protein (Klee and Pimentel-Muinos, 2005), kindly provided by FX Pimentel-Muinos (Instituto de Biología Molecular y Celular del Cáncer, Centro de Investigación del Cáncer, Salamanca, Spain). Detailed information on the conditions used in this study is included in Supplementary data.
Quantitation of apoptotic cells was determined by flow cytometry as the percentage of cells in the sub-G1 region (hypodiploidy) in cell-cycle analysis as previously described (Gajate et al., 2000b).
[Ca2+]ER measurements with aequorin
The BxPC-3 cells were transiently transfected with ER-targeted aequorin (Montero et al., 1997a; Alvarez and Montero, 2002). The cells were plated onto 13-mm round coverslips. Before reconstituting aequorin, [Ca2+]ER was reduced by incubating the cells for 5–10 min at room temperature with the sarcoplasmic and ER Ca2+-ATPase inhibitor 2,5-di-tert-butyl-benzohydroquinone (10 μM) in standard external medium containing: 145 mM NaCl, 5 mM KCl, 1 mM MgCl2, 10 mM glucose and 10 mM HEPES at pH 7.4, supplemented with 3 mM EGTA. Cells were then incubated for 90 min at room temperature in standard medium containing 0.5 mM EGTA, 10 μM 2,5-di-tert-butyl-benzohydroquinone and 2 μM coelenterazine n. The coverslip was then placed in the perfusion chamber of a purpose-built thermostatized luminometer, and standard medium containing 1 mM Ca2+ was perfused to refill the ER with Ca2+. Calibration of the luminescence data into [Ca2+] was made using an algorithm as previously described (Alvarez and Montero, 2002).
Cells (4–5 × 106) were lysed with 60 μl of 25 mM Hepes (pH 7.7), 0.3 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.1% Triton X-100, 20 mM β-glycerophosphate and 0.1 mM sodium orthovanadate, supplemented with protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 20 μg/ml aprotinin and 20 μg/ml leupeptin). Proteins (45 μg) were run on SDS–polyacrylamide gels, transferred to nitrocellulose filters, blocked with 5% (w/v) defatted powder milk in 50 mM Tris–HCl (pH 8.0), 150 mM NaCl and 0.1% Tween 20 for 60 min at room temperature, and then incubated for 1 h at room temperature or overnight at 4 °C with specific antibodies. Detailed information on the antibodies used is included in Supplementary data.
Mitochondrial cytochrome c release measurement
Solid-phase JNK assay
Protein kinase assays were carried out using a fusion protein between glutathione S-transferase and c-Jun (amino acids 1–223) as a substrate of JNK, as previously described (Gajate et al., 1998, 2000a).
Reverse transcriptase–PCR and restriction analysis
Detailed information on the conditions for reverse transcriptase–PCR and primer sequences are included in Supplementary data. Subsequent PstI restriction analysis of the XBP1 amplicon was carried out following standard procedures.
Xenograft mouse models
Female CB17-severe combined immunodeficient mice (8-week old) (Charles River Laboratories, Lyon, France) kept and handled according to institutional guidelines, complying with Spanish legislation under 12/12 h light/dark cycle at a temperature of 22 °C, received a standard diet and acidified water ad libitum. Capan-2 and HuP-T4 cells (5 × 106) were injected subcutaneously in 100 μl phosphate-buffered saline together with 100 μl Matrigel basement membrane matrix (Becton Dickinson, Franklin Lakes, NJ, USA) into the right flank of each mouse. When tumors were palpable, approximately 2 weeks after tumor cell implantation, mice were randomly assigned to cohorts of eight mice each, receiving a daily oral administration of edelfosine (30 mg/kg of body weight) or an equal volume of vehicle (water). The shortest and longest diameter of the tumor were measured with calipers at the indicated time intervals, and tumor volume (mm3) was calculated using the following standard formula: (the shortest diameter)2 × (the longest diameter) × 0.5. Animal body weight and any sign of morbidity were monitored. Drug treatment lasted 32 days for Capan-2-bearing mice and 47 days for HuP-T4-bearing mice. Animals were killed 24 h after the last drug administration according to institutional guidelines, and tumors were carefully removed, weighed and analyzed. A necropsy analysis involving tumors and distinct organs was carried out.
TUNEL assay in tumor sections
The DeadEnd Fluorometric TUNEL System (Promega, Madison, WI, USA) was used to detect apoptosis. Detailed information on the conditions used in this study is included in Supplementary data.
Tumor tissue samples were fixed in 4% buffered paraformaldehyde and embedded in paraffin. Detailed information on the conditions used in this study is included in Supplementary data.
The results given are the mean±s.d. of the number of experiments indicated. Statistical evaluation was performed by Student's t-test. A P-value of <0.05 was considered statistically significant.
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This work was supported by grants from Fondo de Investigación Sanitaria and European Commission (FIS-FEDER PS09/01915), Ministerio de Ciencia e Innovación (SAF2008-02251, BFU 2008-01871 and RD06/0020/1037 from Red Temática de Investigación Cooperativa en Cáncer, Instituto de Salud Carlos III), European Community's Seventh Framework Programme FP7-2007-2013 (Grant HEALTH-F2-2011-256986) and Junta de Castilla y León (CSI052A11-2, GR15-Experimental Therapeutics and Translational Oncology Program, Biomedicine Project 2009 and Biomedicine Project 2010-2011). CG is supported by the Ramón y Cajal Program from the Ministerio de Ciencia e Innovación of Spain.
The authors declare no conflict of interest.
Supplementary Information accompanies the paper on the Oncogene website
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Gajate, C., Matos-da-Silva, M., Dakir, EH. et al. Antitumor alkyl-lysophospholipid analog edelfosine induces apoptosis in pancreatic cancer by targeting endoplasmic reticulum. Oncogene 31, 2627–2639 (2012). https://doi.org/10.1038/onc.2011.446
- endoplasmic reticulum stress
- apoptotic signaling
- pancreatic cancer
- xenograft animal model
- alkyl-lysophospholipid analog
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