PM02734 (Elisidepsin) Induces Caspase-Independent Cell Death Associated with Features of Autophagy, Inhibition of the Akt/mTOR Signaling Pathway, and Activation of Death-Associated Protein Kinase

PM02734 (elisidepsin; Fig. 1A) is a synthetic marine-derived cyclic peptide belonging to the kahalalide family of compounds (1). We and others have previously shown that PM02734 is a potent cytotoxic agent both in vitro and in vivo in human non–small-cell lung cancer (NSCLC) cell lines (2). PM02734 is currently in phase II clinical development with preliminary evidence of antitumor activity. The cellular and molecular mechanisms of PM02734-induced cell death remain to be elucidated.

Type I programmed cell death (apoptosis) is a highly and genetically regulated cell suicide response to facilitate normal embryonic development and homeostasis of multicellular organisms. Apoptotic cell death is characterized by cell shrinkage, chromatin condensation, nucleosomal DNA fragmentation, and formation of apoptotic bodies. Activation of the caspase family of cysteine proteases plays a crucial role in the regulation of apoptosis in the initial and execution stages (3). In addition, apoptosis is an important mechanism of drug-induced tumor cell killing and susceptibility to apoptosis in tumor cells is an important determinant of chemotherapy efficacy (4). Type II programmed cell death, or cell death with autophagy, is characterized by the appearance of double- or multiple-membrane structures called autophagosomes, cytoplasmic vesicles that engulf bulk cytoplasm and/or cytoplasmic organelles such as mitochondria and endoplasmic reticulum. The autophagic vesicles and their contents are degraded by the cellular lysosomal system. Unlike type I programmed cell death, cell death with autophagy is not dependent on the activation of caspases but dependent on the expression of several tumor suppressor genes involved both in the formation of the autophagosomes and the activation of specific signaling pathways (5). Although the role of autophagy as a cell survival or cell death pathway is controversial, a number of studies have described cell death with autophagic features in response to various antitumor agents (6–8). The activation of the class I phosphoinositide 3-kinase (PI3K)/AKT pathway acts as a cell survival signaling pathway. Inhibition of the PI3K/AKT signaling pathway causes cell death associated with apoptosis and/or autophagy (9). The activation of the mTOR signaling pathway, downstream of PI3K/AKT, is implicated in protein synthesis, cell-cycle progression, and cell proliferation (10). Interestingly, inhibition of mTOR signaling has been shown to trigger cell death with autophagy (11), as mTOR acts as a repressor of the autophagic pathway.

Death-associated protein kinase (DAPK) is a calcium/calmodulin-regulated serine/threonine kinase associated with actin cytoskeleton (12). It plays a role as a positive mediator in response to various apoptotic stimuli such as IFN-γ (13), activation of Fas receptors, TNF-α, TGF-β (14), ceramide-, and p53-mediated apoptosis (15), as well as in the disruption of the extracellular matrix and resulting cell detachment. DAPK appears to function as a sensor that triggers early apoptotic events (16). In addition, DAPK acts as a tumor suppressor, as loss of DAPK expression secondary to hypermethylation is involved in tumor formation (17). Recent studies have shown that DAPK is a novel regulator of autophagic signaling in cell death mediated by estrogen receptor (ER) stress (18). DAPK belongs to a family of highly related death-associated kinases, which includes DRP-1 and ZIP kinases (19). DAPK contains a kinase domain, a calmodulin regulatory segment, 8 ankyrin repeats, a cytoskeleton binding region, and a death domain. The activation of DAPK depends on its dephosphorylation at serine 308 in the Ca⁺²/calmodulin regulatory domain in response to cell death stimuli, resulting in its proapoptotic activation (20). In addition, activation of DAPK may have a direct role in the regulation of autophagic signaling pathways (21).

In this article, we show that PM02734 induces cell death associated with autophagic features including an increase in the number of acidic vacuole organelles, due, for the most part, to a remarkable compromise in the clearance of autophagosomes and/or autolysosomes. PM02734 also caused inhibition of AKT/mTOR signaling, and cotreatment with the AKT inhibitor wortmannin or with the mTOR inhibitor rapamycin enhanced PM02734-induced cytotoxicity associated with autophagic features. Furthermore, PM02734 induced the activation of DAPK and downregulation of DAPK gene expression by siRNA led to the attenuation of PM02734-induced cell death. PM02734 exhibited in vivo antitumor activity associated with the activation of the autophagic pathway in nude mice bearing human A549 NSCLC xenografts. Overall, these observations provide novel insight into the complex mechanism of PM02734-induced cell death that relays in part in its effects on the autophagic pathway at the level of steady-state number and clearance of autophagic compartments. Our findings should help building a rational basis for its therapeutic application either alone or in combination with other chemotherapeutic agents.

PM02734 was manufactured by PharmaMar and dissolved in dimethyl sulfoxide (DMSO) as a stock concentration of 10 mmol/L and diluted to the indicated concentrations with culture medium. Polyclonal anti-caspase-8, caspase-9, caspase-3, anti-PARP antibody, anti-AKT, anti-phospho-AKT (Ser437) and anti-phospo-AKT (Thr308), anti-mTOR, and anti-phospho-mTOR (Ser2448), anti-S6 ribosomal protein, and anti-phospho-S6 ribosomal protein (Ser235/236), as well as anti-4E-BP1 antibody and anti-phospho-4E-BP1 (Thr37/46) antibodies were purchased from Cell Signaling Technology. Polyclonal anti-LC3 and anti-Atg-5 antibodies were obtained from Novus Biologicals. Polyclonal p62/SQSTM1 antibody and monoclonal anti-DAPK and anti-phospho-DAPK (Ser308) antibodies were purchased from Sigma-Aldrich. Other chemicals and reagents were obtained from Sigma-Aldrich, or from Biomol.

Human NSCLC cell lines H322 and A549 were obtained from American Type Culture Collection. Cell lines were maintained in RPMI 1640 medium with 10% FBS and maintained at 37°C in a humidified atmosphere of 95% air and 5% CO₂.

Exponentially growing cells (1 × 10⁵/mL) were plated in 96-well plates and allowed to attach overnight. Cells were exposed to various concentrations of PM02734 for the indicated times. Cell viability was assessed by the reduction of tetrazolium bromide (MTT) assay. For assay of cell death, cells were treated with PM02734 at different concentrations for the indicated times. After treatment, cell death was determined by trypan blue exclusion as described by Scarlatti and colleagues (22).

Cells were treated with 0.5 to 1 μmol/L PM02734 for 6 hours, and then cell extracts were prepared with extraction buffer. Ten microliter of cell extracts (10–30 μg of protein) were added into 100 μL of reaction mixture containing 12 μmol/L Ac-DEVD-pNA (Biomol) as the substrate for caspase-3 in a 96-well microplate. After incubation at room temperature for 120 minutes, the amount of p-nitroaniline–derived substrate cleavage by caspase-3 was determined in a microplate reader (Molecular Devices) at 405 nm.

Cells were treated with 0.5 to 1 μmol/L PM02734 for the indicated times. After treatment, cells were washed twice with PBS solution and then stained with 1 μg/mL acridine orange solution (Invitrogen) at 37°C for 15 minutes. The levels of acidic vesicular organelles (AVO) were assessed by counting the number of bright red fluorescent cells from a total of at least 200 cells with a Nikon fluorescence microscope.

GFP-LC3 plasmid was a gift from Dr. Mizushima (Department of Bioregulation and Metabolism, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan). Cells grown at about 70% confluence were transiently transfected with 1 μg/mL of GFP-LC3 plasmid by using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. The localization of GFP-LC3 in transfected cells was assessed by fluorescence microscopy. For downregulation of Atg-5 and DAPK gene expression, Atg-5 siRNA and DAPK siRNA were purchased from Dharmacon. Transfections of siRNA were carried out with Lipofectamine 2000 according to the manufacturer's instructions.

A549 cells were treated with 1 μmol/L PM02734 or with the same volume of medium containing 0.1% DMSO as a control at 37°C for 8 hours. After treatment, cells were washed 3 times with PBS, and then fixed with 0.5 mL of ice-cold glutaraldehyde (2.5% in 0.1 mol/L cacodylate buffer, pH 7.4) at 4°C overnight. After washing, cells were fixed in 1% OsO₄ and embedded in polybed resin. The ultrathin sections were doubly stained with uranyl acetate and lead citrate and analyzed by transmission electron microscopy (JEOL).

Cells were scraped from culture dishes, and cell lysates were prepared with lysis buffer. Immunoblot analysis was conducted as previously described (23). Autophagic flux was calculated from the densitometric analysis of the immunoblots for LC3, as the ratio of LC3-II levels in cells in the presence of bafilomycin over untreated cells (LC3 flux), or the amount of LC3-II in bafilomycin-treated cells minus that in untreated cells (LC3 net flux; ref. 24).

Endogenous DAPK was immunoprecipitated from PM02734-treated A549 cells at the indicated times and subjected to an in vitro kinase assay using myosin II regulator light chain (MLC; Sigma-Aldrich) as a substrate as described by Jin and colleagues (25). DAPK activity was assessed by measurement of phosphorylated MLC by immunoblot analysis using anti-phos-MLC antibody.

Exponentially growing A549 cells (2 × 10⁶ cells) were subcutaneously injected into the flank of nude mice. Seven days after inoculation mice bearing tumors (volume around 100 mm³) were divided into 2 groups of 3 animals and treated with 0.1 mg/kg PM02734 or the same volume of vehicle as control, via tail vein 3 times per week for 2 weeks. Body weight and tumor size were monitored at the indicated time points. All animal experiments were conducted in accordance with Institutional Protocol for Animal Experiments of the Albert Einstein College of Medicine. After 2 weeks, mice were sacrificed, and tumor tissues from control and PM02734-treated mice were harvested for assay of apoptosis by terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) staining or for assay of autophagy as measurement of the levels of LC3-II by either immunohistochemical staining or by immunoblotting using a polyclonal anti-LC3 antibody (NB600-1384; Novus Biologicals).

All data are presented as mean ± SD. Differences between groups were analyzed for statistical significance using a 2-tailed Student's t test. P < 0.05 was used as the significance level.

We have previously studied the cytotoxic effect of PM02734 on a panel of human NSCLC cell lines and observed that H322 and A549 cell lines are highly sensitive to PM02734 with IC₅₀ values of 0.5 to 1 μmol/L (2). Initially, we treated H322 and A549 cells with varying concentrations of PM02734 for 8 hours or with 0.5 μmol/L PM02734 (H322 cells) or 1 μmol/L PM02734 (A549 cells) for the indicated times to evaluate drug-induced cell death as measured by trypan blue exclusion. As shown in Figure 1B, PM02734 induces cell death in a concentration- and time-dependent manner, about 12% to 17% cell death can be detected at 4 hours after drug exposure and 37% to 40% at 24 hours. By morphologic assessment, PM02734 induced disruption of membrane integrity and nuclear condensation but did not cause chromatin fragmentation (Fig. 1C), suggesting that cell death induced by PM02734 does not have the characteristics of classical apoptosis. To further verify drug-induced membrane disruption, we treated A549 cells with varying concentrations of PM02734 for 6 hours and then stained them with fluorescein isothiocyanate (FITC)-labeled Annexin V. PM02734 treatment caused FITC-labeled Annexin V–positive staining cells with exposure of phosphatidylserine outside the plasma membrane occurring at 1 μmol/L and increasing at 10 μmol/L (data not shown). We next explored whether PM02734-induced cell death was associated with caspase activation. Immunoblot analysis revealed that PM02734 treatment for 8 hours did not markedly induce the formation of the active forms of cleaved caspase-8, capase-9, and caspase-3, whereas the active forms of caspase-8 (43 and 41 kDa), caspase-9 (39 and 37 kDa), and caspase-3 (28, 19, and 17) were clearly observed in the tested cell lines following treatment with 1 μmol/L staurosporine. Consistently, the cleavage of PARP, which is a substrate of caspase-3 and a well-known apoptotic hallmark, was observed only in cells treated with staurosporine but not in PM02734-treated cells (Fig. 1D, left). Quantitative analysis of caspase-3 activity by colorimetric assay also showed that PM02734 treatment did not alter caspase-3 activity in the tested cell lines, whereas staurosporine treatment led to an increase in caspase-3 activity (data not shown). Moreover, cotreatment with 50 μmol/L Z-VAD-fmk, a pan caspase inhibitor, failed to inhibit PM02734-induced A549 cell death but effectively reduced staurosporine-induced A549 cell death (Fig. 1D, right). These data suggest that PM02734-induced cell death is not associated with the caspase-dependent apoptotic signaling cascade.

Because PM0274-induced cell death appeared to lack the characteristics of classical apoptotic type I cell death, we sought to determine whether it was associated with changes in the autophagic pathway previously described as characteristics of type II cell death. We first determined the effect of PM02734 on the levels of AVOs by staining tested cells with acridine orange solution as described by Paglin and colleagues (26). Figure 2A, left, shows acidic vesicle organelles in both tested cell lines following treatment with PM02734 for 8 hours, the cytoplasm fluorescence changing from bright green to bright red. Quantitative analysis showed about 60% to 66% cells with an expansion of their AVOs as compared with 15% to 23% in control cells (P < 0.01; Fig. 2A, right). These results support that treatment with PM02734 results in enlargement of the endosomal/lysosomal system in cancer cells. To determine the possible autophagic nature of the PM02734-induced compartments, next, we determined the effect of the drug on the intracellular localization of microtubule-associated protein 1 light chain 3 (LC3), a specific marker of autophagosomes (27), in the tested cell lines. Initially, we transiently transfected cells with GFP-LC3 plasmid for 36 hours and then treated them with PM02734, or with the same volume of medium containing 0.1% DMSO as a control, for an additional 8 hours. The cellular localization of GFP-LC3 protein was assessed by fluorescence microscopy. As shown in Figure 2B, left, representative fluorescence micrographs show a punctate pattern of GFP-LC3 in PM02734-treated cells in contrast to the diffuse pattern in control cells. Quantitative analysis revealed a significant increase in cells with GFP-LC3 punctate pattern in PM02734-treated cells as compared with control cells (P < 0.01), that is, ∼38% versus ∼20% in H322 cells and ∼36% versus ∼17% in A549 cells, respectively (right). To further verify an increase in autophagic markers induced by PM02734, we assessed the effect of PM02734 on the levels of Atg-5/12, a protein complex formed by activation of autophagy (28), and on the conversion of the cytoplasmic form of LC3-I protein (18 kDa) to the preautophagosomal and autophagosomal membrane–bound form of LC3-II (16 kDa). As shown in Figure 2C, left, the immunoblot analysis revealed that PM02734 treatment led to a time-dependent increase in the levels of conjugated Atg-5 and LC3-II proteins in both tested cell lines. Quantitative analysis indicated that PM02734 treatment led to an increase in Atg-5/12 and LC3-II levels as early as 4 hours after drug exposure and gradually increasing thereafter (Fig. 2C, right). To determine whether PM02734-induced accumulation of LC3-II is caused by enhanced formation of autophagic vacuoles or to blockade of autophagic vacuole clearance, we treated cells with PM02734 in the absence or presence of the inhibitor of lysosomal proteolysis bafilomycin A1 (BFA; ref. 24). As shown in Figure 2D (top), LC3-II levels were increased in PM02734-treated cells compared with that in control cells both in the presence and in the absence of bafilomycin. However, measurement of the increase in LC3-II after addition of bafilomycin (which reflects the amount of autophagosomes degraded in lysosomes) revealed that differences between PM02734-treated cells and untreated cells were no longer evident (Fig. 2D). These results support that the increase in the amount of autophagosomes was in large part resulting from their compromised clearance by lysosomes. Reduced autophagosome clearance was also supported when following the degradation of p62/SQSTM1, a polyubiquitin-binding protein that is degraded by autophagy (29, 30). Although levels of p62 were markedly reduced in H322 and A549 cells treated with PM02734, the difference was not due to increased degradation as the lower p62 content in treated cells was still evident upon bafilomycin blockade of lysosomal degradation (Fig. 2D); however, the fact that even after treatment of PM02734, we found a slight but consistent increase in p62 levels in bafilomycin-treated cells supports that some level of autophagosome/lysosome fusion still occurs in these cells. These data suggest that PM02734 may lead to increase content of cellular autophagosomes (increased Atg-5/12 levels) by preventing their clearance (reduced autophagic flux; Fig. 2D, bottom). Finally, we confirmed the increased content of autophagic vacuoles by transmission electron microscopy in A549 cells following treatment with 1 μmol/L PM02734, or with the same volume of medium containing 0.1% DMSO as a control, for 8 hours. As shown in Figure 2E, treatment with PM02734 caused the accumulation of autophagic vacuoles, which exhibited autophagosome and/or autolysosomal characteristics (double and multilayer membranes containing identifiable mitochondrial and cytoplasmic components), whereas only a few vacuoles were observed in control cells. Overall, these results suggest that PM02734-induced cell death may result, at least in part, from the abnormal accumulation of autophagic compartments, which has been shown to be toxic for cells in different cellular settings (31).

To confirm the contribution of the increase in the autophagic compartment to PM02734-induced cell death, we analyzed the effect of 3-methyladenine (3-MA), a well-known inhibitor of autophagosome formation (32). First, we cotreated H322 and A549 cells with 2 mmol/L 3-MA and 0.5 to 1 μmol/L PM02734 for 8 hours, and then harvested cell pellets to determine the amount of the autophagic marker LC3-II. As expected, PM02734 treatment resulted in increased levels of LC3-II in H322 and A549 cells. 3-MA treatment led to a significant decrease in PM02734-induced LC3-II expression in the 2 cell lines (Fig. 3A and B, top and middle). In addition, we tested whether cotreatment with 3-MA could affect PM02734-induced cell death. As shown in Figure 3A and B, bottom, cotreatment with 2 mmol/L 3-MA resulted in a significant but partial inhibition of PM02734-induced cell death, as measured by MTT assay, in H322 and A549 cell lines. Given that Atg-5 protein is essential for induction of autophagy (33), we next determined whether the downregulation of Atg-5 gene expression by transfection with Atg-5 siRNA could affect PM02734-induced cell death. As shown in Figure 3C and D, downregulation of Atg-5 gene expression produced a significant although partial attenuation of PM02734-induced cell death as measured by MTT assay in H322 and A549 cells. The fact that both chemical and genetic blockage of autophagosome formation reduced cell death confirmed that the observed accumulation of autophagosomes in cells treated with this drug contributes at least, in part, to its cellular toxicity.

Activation of the Akt signaling pathway has been shown to be involved in cell survival through phosphorylation at Thr308 by PDK1 or at Ser473 by mTOR kinase (34). mTOR, one of downstream targets of Akt, plays a central role in the regulation of protein synthesis by phosphorylation of p70 S6 kinase and 4E-BP1 (35). Recent investigations have showed that the inhibition of this signaling pathway is linked to the triggering of autophagy (36). Thus, we sought to test whether PM02734 could induce autophagy by inhibition of the Akt/mTOR signaling pathways. As shown in Figure 4A, PM02734 treatment caused a marked reduction in Akt phosphorylation at Ser473 and Thr308, along with the inhibition of phosphorylation of mTOR at Ser2448, as early as 4 hours after exposure and increasing thereafter. In parallel, PM02734 inhibited the phosphorylation of S6 ribosomal protein and 4E-BP1, which are the downstream targets in response to Akt/mTOR signaling. Of interest, 4E-BP1 appeared to be more susceptible to PM02734 as strong inhibition of 4E-BP1 phosphorylation was already observed at 2 hours of drug exposure. To further explore the role of Akt/mTOR signaling inhibition in the regulation of PM02734-induced cell death, we cotreated H322 and A549 cells with PM02734 and the Akt inhibitor wortmannin or the mTOR inhibitor rapamycin for 24 hours. Following treatment, cell viability was determined by MTT assay, and the LC3-II levels were assayed by immunoblotting. As shown in Figure 4B, either cotreatment with wortmannin or with rapamycin resulted in a significant enhancement of cell death as compared with PM02734 alone (P < 0.05–0.01). Although wortmannin has been shown to inhibit PI3K class III and compromise autophagosome formation, in a similar manner to previously shown for 3MA, this did not seem to be the case in these cells in the conditions of our assay. In fact, the immunoblot analysis revealed an increased amount of LC3-II in these cells when treated with wortmannin almost comparable to the one induced by rapamycin. Addition of PM02734 further increased LC3-II levels in cells treated with either wortmannin or rapamycin, further supporting that the effect of PM02734 was not only at the level of autophagosome formation but also on autophagosome clearance (Fig. 4C).

To further test whether cotreatment with Akt and mTOR inhibitors could affect PM02734-induced autophagic flux, we also determined the intracellular levels of p62 protein. As shown in Fig. 4D, PM02734 markedly suppressed p62 expression as compared with that in control cells. Treatment with Akt inhibitor wortmannin alone led to a remarkable accumulation of p62 protein; however, treatment with mTOR inhibitor rapamycin caused an effective decrease in p62 protein. Of interest, cotreatment with PM02734 resulted in a significant suppression of p62 levels as compared with wortmannin or rapamycin alone (P < 0.01). These results suggest that the inactivation of Akt/mTOR pathway by PM02734 may be involved in the regulation of drug-induced autophagy and cell death.

Recent studies have shown that the activation of DAPK is associated with the triggering of types I and II cell death (5, 19, 37). Thus, we wanted to test whether PM02734-induced cell death could be associated with the activation of DAPK in A549 cells. Initially, we tested the effect of PM02734 on DAPK expression and found that PM02734 treatment caused the degradation of full-size DAPK (160 kDa) accompanied by increased levels of its corresponding cleaved fragments (100 and 60 kDa) in a concentration- and time-dependent manner (Fig. 5A). Although the significance of DAPK degradation remains largely unknown, several studies have shown that the generation of proteolytic DAPK fragments (100 or 60 kDa) is involved in the initiation of the apoptotic and autophagic cascades (20, 38). Furthermore, recent investigations have shown that the activation of DAPK depends on its dephosphorylation at Ser308 (20). We therefore determined the effect of PM02734 on DAPK phosphorylation at Ser308 by immunoblot analysis using the corresponding antibody. Of interest, PM02734 treatment strongly induced the dephosphorylation of DAPK at Ser308 in a concentration- and time-dependent manner. As shown in Figure 5B, left, 1 μmol/L PM02734 led to about 80% of DAPK dephosphorylation at Ser308 in A549 cells. Time course studies showed about 65% DAPK dephosphorylation as early as 1 hour after PM02734 exposure and about 80% DAPK dephosphorylation during the 4- to 24-hour period after drug exposure (Fig. 5B, right). To further confirm that PM02734-induced DAPK dephosphorylation was correlated with the enhancement of DAPK activity, we used MLC as a substrate to determine the effect of PM02734 on DAPK activity in vitro using immunoprecipitated endogenous DAPK as described in Materials and Methods. As shown in Figure 5C, DAPK activity was increased by about 150% as early as 1 hour of drug exposure and reached its maximum (280% to 250%) after 2 to 4 hours of exposure, declining to basal levels after 24-hour exposure. Moreover, we determined the effect of downregulation of DAPK expression on PM02734-induced cell death. As shown in Figure 5D, downregulation of DAPK expression by siRNA resulted in a pronounced and significant attenuation of PM02734-induced cell growth inhibition as measured by MTT assay. In addition, downregulation of DAPK by siRNA transfection also resulted in a significant although incomplete attenuation of PM02734-induced increase in LC3-II levels in A549 cells (Fig. 5E). These results suggest a relationship between PM02734-induced activation of DAPK and autophagy.

To investigate the antitumor efficacy of PM02734 in vivo, we conducted a dose escalation toxicity trial in mice and found that an intravenous dose of 0.1 mg/kg was well tolerated. We then established a xenograft model of human A549 NSCLC cells in nude mice. Exponentially growing A549 cells (2 × 10⁶ cells) were subcutaneously inoculated in the flank of nude mice. Seven days after inoculation, when tumor volume is about 100 mm³, mice were randomly assigned to control (vehicle) and PM02734 treatment groups. Mice bearing established A549 tumors were given intravenous injections of 0.1 mg/kg PM02734 or the same volume of vehicle 3 times per week for 2 weeks. As shown in Figure 6A, top, PM02734 caused about 50% tumor growth inhibition (P < 0.05) as compared with control animals. No body weight loss was observed in drug-treated mice compared with control mice (Fig. 6A, bottom). To investigate the mechanism of PM02734-induced tumor growth inhibition in vivo, mice were sacrificed and tumor tissues collected 1 day after the last PM02734 dose. Apoptosis was assessed by TUNEL staining and autophagy by LC3 expression by immunohistochemical staining and immunoblot analysis. Figure 6B shows no differences in the fraction of TUNEL-positive staining cells between PM02734-treated tumors and control tumors. In contrast, the expression of total cytoplasmic LC3 detected by immunohistochemical staining was markedly elevated in PM02734 treated tumors compared with control tumors (Fig. 6C). Consistently, the immunoblot analysis revealed that LC3-II levels were higher in PM02734-treated tumors than in control tumors, whereas no differences were observed between PM02734 treatment and control tumors in Bcl-2 and Bax levels (Fig. 6D). All these data suggest that autophagosome accumulation is associated with PM02734-induced tumor growth inhibition in vivo.

Our results indicate that PM02734-induced cell death is associated with features of autophagy including an increase in cellular content of AVOs and compromised autophagic clearance. The role of autophagy in cancer therapeutics is still controversial. Autophagy has been found to play a role as a cell survival mechanism by which cells clear damaged cytoplasmic proteins and organelles through lysosomal degradation and survive metabolic stress (39). On the other hand, autophagy has been also found to contribute to type II programmed cell death in response to hypoxia, chemotherapeutic agents, virus infection, and toxins (40). Our results suggest that the observed abnormal increase of the autophagic compartment can only partially explain the cytotoxic effect of PM02734, as cotreatment with 3-MA, a well-known autophagy inhibitor, or Agt-5 siRNA, an essential component for autophagosome formation, had only a partial protective effect on PM02734-induced cell death in H322 and A549 cells. It is possible that the PM02734-induced increase in autophagosome content could be, in part, related to induction of autophagy through its inhibitory effect on the Akt/mTOR signaling pathway and activation of DAPK. The accumulation of autophagic vacuoles induced by PM02734 was confirmed in vivo in A549 xenografts. This study is the first to show that PM02734-induced antitumor effect is, in part, mediated by an autophagic mechanism.

Apoptosis is an active form of cell death characterized by cell shrinking, cytoplasmic condensation, DNA ladder degradation, and nuclear fragmentation resulting in the formation of apoptotic bodies. In contrast, type II programmed cell death is characterized by the presence of visible autophagic cytoplasmic vacuoles, mitochondrial dilation, enlargement of the endoplasmic reticulum and the Golgi apparatus, and nuclear condensation without DNA laddering (41). Apoptosis and cell death with autophagy are not mutually exclusive and can coincide in vivo in certain tissues and in cell culture (42). In addition, apoptosis and autophagy may share certain mechanisms and alterations such as loss of mitochondrial permeability and membrane potential (43). The fact that PM02734-induced cell death could only be attenuated partially with autophagy inhibitors suggests that other mechanisms of cell death are involved in PM02734-induced caspase-independent cell death. In a previous work, we and others have shown that kahalalide F, a parent compound of PM02734, induces necrotic cell death (type III programmed cell death) linked to damage of the endoplasmic reticulum, Golgi apparatus, and the activation of lysosomes, as well as loss of the mitochondrial membrane potential and release of lactate dehydrogenase (LDH) from cytoplasm (44). Preliminary studies also show that PM02734 leads to a rapid depletion of intracellular ATP and loss of mitochondrial membrane potential in H322 and A549 cells (data not shown), suggesting that activation of necrotic signaling pathways may also be involved in PM02374-induced cell death.

An emerging body of evidence indicates that the Akt/mTOR pathway plays a crucial role in the regulation of both apoptosis and autophagy (36). We present here data indicating that PM02734 markedly inhibits Akt phosphorylation at Ser473 and at Thr308, as well as mTOR phosphorylation at Ser2448. In addition, we observed that downregulation of mTOR protein by PM02734 may explain at least in part mTOR dephosphorylation. Time course studies indicated that the inhibition of the Akt/mTOR signaling pathway occurred as early as 4 hours after PM02734 exposure, suggesting that the inhibition of Akt/mTOR is an early event in the drug-induced cell death process. As expected, phosphorylation of S6 ribosomal protein and 4E-BP-1, downstream targets of Akt/mTOR pathway, were inhibited by PM02734. However, we found that PM02734 caused degradation and dephosphorylation of 4E-BP-1, a translation factor for protein synthesis and a downstream element of the Akt/mTOR axis 2 hours after PM02734 exposure, suggesting that inactivation of the S6 ribosomal protein/4E-BP1 pathway preceded Akt/mTOR inhibition. Although the pharmacologic significance of PM02734-induced downregulation and inactivation of Akt/mTOR downstream events is unknown, these findings suggest that the effect of PM02734 on this pathway could be 2-fold; in one hand, it may induce autophagosome formation, which associated with the observed compromise in clearance would lead to cellular clogging, and in addition, it may directly or indirectly affect protein biosynthesis, which could further lead to suppression of cell proliferation and cell growth. Wortmannin has been shown to be an autophagy inhibitor via inhibition of class III PI3K (45); however, its effect over class I PI3K would result in mTOR inhibition. In this work, we found that wortmannin markedly induces accumulation of the intracellular levels of LC3-II in H322 and A549 cells, indicating that this compound may have a preferential effect on class I PI3K in these cells, as it did not seem to impact the conversion of LC3-I into LC3-II, or could be interfering with autophagosome clearance by lysosomes. Recent investigations have shown that autophagy inhibition at an early stage could trigger cell survival signaling pathways, preventing death due to stress; whereas, inhibition of autophagy at a late stage could cause accelerated cell death under autophagy-inducing conditions via the activation of apoptotic signaling (46). Further studies are required to determine whether a combined effect of PM02734 activating early autophagy steps and compromising late autophagic stages may be behind its marked negative effect on cell viability. Interestingly, we here present data that cotreatment with 3-MA, which is an early autophagy inhibitor (45), led to the attenuation of PM02734-induced cell death, whereas cotreatment with wortmannin, which may inhibit autophagy at a late stage as discussed above, caused a significant enhancement of PM02734-induced cytotoxicity in 2 tested cell lines. Consistent with our findings, Shingu and colleagues recently reported that inhibition of autophagy by 3-MA attenuated imatinib-induced cell death, but inhibition of autophagy at a late stage with bafilomycin A1 enhanced drug-induced cytotoxicity in human malignant glioma cells (47). In addition, we also observed that cotreatment with the mTOR inhibitor rapamycin and PM02734 resulted in an additive effect on cell death associated with an increase in autophagosome content as shown by increase in LC3-II, suggesting that the inhibition of mTOR signaling may play a role in PM02734-induced autophagy. Overall, the role of the PI3K/Akt/mTOR axis in modulating PM02734-induced cell death appears highly complex and deserves further investigation.

DAPK, Ca²⁺/calmodulin-regulated serine/threonine kinases, has a wide range of functions in the regulation of cell growth and cell death. It functions as a tumor suppressor, is involved in p53-mediated checkpoint control resulting in inhibition of oncogene-induced cell transformation, and is also an antimetastasis factor (12). Recent studies have shown that DAPK acts as a positive mediator, regulating different types of cell death signaling pathways including apoptosis and autophagy (18, 21). Of note, data presented in this work indicate that PM02734 induces the cleavage of full-size DAPK (160 kDa) into 2 small fragments (100 and 60 kDa) in a dose- and time-dependent manner. The biological role of such cleavage is unclear, although Shamloo and colleagues (38) have reported that the cleavage of full-size DAPK resulting in 100 and 60 kDa fragments was correlated with the induction of cell death in damaged rat brain by ischemic injury, suggesting that these cleaved fragments may represent the activated form of DAPK. Moreover, a number of investigations have showed that the activation of DAPK depends on its dephosphorylation at Ser308 (21). Consistently, we also found that PM02734 at a low concentration (1 μmol/L) induces DAPK dephosphorylation at Ser308 as early as 1 hour after exposure, and an increase in DAPK dephosphorylation over time. Accordingly, DAPK activity was induced after PM02734 exposure for 1 hour, peaked at 4 to 8 hours and declined thereafter, suggesting that the activation of DAPK may be an initiator of cell death. Of interest, downregulation of DAPK expression by siRNA caused a significant attenuation of PM02734-induced cytotoxicity and also blocked drug-induced LC3-II accumulation, suggesting that the activation of DAPK may contribute to PM02734-induced cell death with autophagic features. Recently, Harrison and colleagues reported that DAPK can interact with microtubule-associated protein 1B, which was proven to interact with autophagosomal protein Atg-8 (LC3), resulting in the stimulation of DAPK-dependent membrane blebbing and autophagy (48). In addition, Zalckvar and colleagues showed that DAPK phosphorylates beclin 1, an essential protein for autophagy at threonine 119, resulting in the dissociation of beclin 1 from Bcl-XL and induction of autophagy (49). In addition, we here observed that downregulation of DAPK by siRNA causes greater extent of blockade of PM02734-induced cell death than direct inhibition of autophagy by 3-MA treatment or by Atg-5 downregulation, suggesting that other lethal caspase-independent pathways induced by DAPK such as activation of programmed necrosis may be involved in PM02734-induced cell death. Thus, the cellular and molecular mechanism by which PM02734 induces DAPK activation and cell death associated with autophagic features deserves to be studied further. Consistent with the in vitro results, we found that PM02374 has antitumor activity in vivo and that the extent of LC3-II, an autophagic marker, was elevated in PM02734-treated xenograft tumor tissues as measured by immunohistochemical staining and immunoblot analysis. However, TUNEL assay and the levels of Bcl-2 and Bax did not show evidence of apoptosis after PM02734 treatment.

In summary, we have shown that PM02734 induces cell death in human NSCLC cell lines both in vitro and in vivo by a complex mechanism that appears to involve accumulation of autophagosomes, mainly by compromise of autophagosome clearance along with inhibition of the Akt/mTOR signaling pathway and activation of DAPK. Consistent with in vitro results, PM02734 inhibits tumor growth accompanied by marked accumulation of autophagosomes in vivo in A549 xenografts. These observations represent a potentially first important step toward understanding the mechanism of lethality of this structurally unique agent and may be of help in rationally integrating PM02734 with other agents for the treatment of NSCLC patients.

No potential conflicts of interest were disclosed.

This work was supported in part by NIH grants CA84119 and CA96515 and by PharmaMar R & D.

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