Stellettin B Induces G1 Arrest, Apoptosis and Autophagy in Human Non-small Cell Lung Cancer A549 Cells via Blocking PI3K/Akt/mTOR Pathway

Until now, there is not yet antitumor drug with dramatically improved efficacy on non-small cell lung cancer (NSCLC). Marine organisms are rich source of novel compounds with various activities. We isolated stellettin B (Stel B) from marine sponge Jaspis stellifera, and demonstrated that it induced G1 arrest, apoptosis and autophagy at low concentrations in human NSCLC A549 cells. G1 arrest by Stel B might be attributed to the reduction of cyclin D1 and enhancement of p27 expression. The apoptosis induction might be related to the cleavage of PARP and increase of ROS generation. Moreover, we demonstrated that Stel B induced autophagy in A549 cells by use of various assays including monodansylcadaverine (MDC) staining, transmission electron microscopy (TEM), tandem mRFP-GFP-LC3 fluorescence microscopy, and western blot detection of the autophagy markers of LC3B, p62 and Atg5. Meanwhile, Stel B inhibited the expression of PI3K-p110, and the phosphorylation of PDK1, Akt, mTOR, p70S6K as well as GSK-3β, suggesting the correlation of blocking PI3K/Akt/mTOR pathway with the above antitumor activities. Together, our findings indicate the antitumor potential of Stel B for NSCLC by targeting PI3K/Akt/mTOR pathway.

In this paper we report the antitumor effects of Stel B on human NSCLC A549 cells, including the cell cycle G1 arrest, apoptosis and autophagy induction.

Antiproliferative effect of Stel B on human NSCLC A549 cells. The antiproliferative activity of Stel B
on A549 cells was investigated by WST-8 assay, a sensitive colorimetric assay to determine cell viability by measuring dehydrogenase in living cells. After treatment by Stel B for 48 h, proliferation of A549 cells was inhibited in a dose-dependent manner (Fig. 1), with the IC50 value as 0.022 μ M, suggesting the high inhibitory potency of Stel B. In addition, Stel B inhibited A549 proliferation time-dependently, with treatment for 72 h or longer at 5 μ M leading to 100% inhibition (supplementary Fig. S1). And we preliminarily investigated whether A549 cells can develop resistance to Stel B. As a result, no obvious resistance occurred after ten days of exposure to 0.002 μ M Stel B (supplementary Fig. S2).

Stel B induced cell cycle arrest at G1 phase in A549 cells.
Cell cycle is a repeating series of events that take place in a cell leading to its division and DNA replication to produce two daughter cells. Disturbance of cancer cell cycle would inhibit cell growth and activate apoptosis process. To examine whether Stel B affects cell cycle in A549 cells, we examined the cell cycle distribution after treatment with Stel B for 24 h. As shown in Fig. 2a, a dose-dependent accumulation of cells in G1 phase was observed after Stel B treatment. In contrast, the percentage of cells in S and G2/M phases reduced as Stel B concentration increased (Fig. 2b).

Effect of Stel B on the expression of cell cycle-related proteins.
Cell cycle is known to be regulated positively by cyclin-CDK (cyclin dependent kinase) complexes, but negatively by CDK inhibitors such as p27. To explore the potential molecular mechanism by which Stel B caused G1 arrest, we analyzed the expression levels in total and nuclear protein of the molecules which are known to be involved in G1/S checkpoint. Figure 2c and supplementary Fig. S4 show that Stel B significantly reduced the expression of cyclin D1 and phosphorylation of pRB, and increased the expression of CDK inhibitor p27, in nucleus of A549 cells. The level of cyclin D1 in the whole cell was downregulated as well. These results suggest that the G1 arrest induced by Stel B might be attributed to the effect on cyclin D1, p27 as well as pRb.

Stel B induced apoptosis in A549 cells.
To investigate whether Stel B induced apoptosis in A549 cells, we used Annexin V-FITC/PI double staining to measure the population of apoptotic cells. The population at upper-right quadrate (Annexin V + /PI + ) increased to 8.34%, 12.0%, 15.8% and 27.6%, respectively, after treatment with 0.02, 0.05, 0.25, 1 μ M of Stel B, compared to that for control cells (5.86%), suggesting that treatment with Stel B induced late-stage apoptosis in A549 cells (Fig. 3a).
To demonstrate the apoptotic induction, microscopy with DAPI staining was carried out. As shown in Fig. 3b, cytoplasmic shrinkage and nuclear fragmentation occurred after treatment with 1 μ M of Stel B or 5 μ M of Adriamycin (ADR), supporting that apoptosis was indeed induced.
As an important signaling protein involved in DNA repair and apoptosis, PARP is cleaved by upstream caspases when apoptosis occurs. Therefore, cleavage of PARP is widely used as a marker of apoptosis. We Stel B increased reactive oxygen species (ROS) production. By changing the internal environment of cells, ROS accumulation is known to play an important role in apoptosis 16 . We used DCFH-DA as a molecular probe to detect intracellular ROS production. As indicated in Fig. 4, the ROS level increased in a concentration-dependent manner following Stel B treatment, suggesting that Stel B promoted ROS generation in A549 cells.

Stel B induced autophagy in A549 cells.
Autophagy is a lysosomal degradation process for cytoplasmic constituents during the stress condition. To examine whether Stel B induced autophagy in A549 cells, several autophagic assays were performed. First, we used monodansylcadaverine (MDC) staining to detect autophagic vacuoles. As shown in Fig. 5a, Stel B induced the accumulation of MDC-labeled vacuoles in the cytoplasm in a dose-dependent manner. Rapamycin (Rapa), a well-known autophagy inducer, also obviously increased MDC-staining puncta production.
To elucidate cytological changes in autophagic cells induced by Stel B, we examined the intracellular morphologic change of A549 cells by use of transmission electron microscopy (TEM). As shown in the electron micrographs, a 450% increase of autophagic vacuoles which contain subcellular materials, were observed in Stel B-treated cells, compared with those in control cells (Fig. 5b). The size of autophagic vacuoles containing remnants of organelles was approximately 1 μ m in diameter.
Moreover, we determined the effect of Stel B on the expression of autophagy marker proteins including LC3B, p62 and Atg5 by western blot. Amount of LC3B II/I was reported to be proportional to the number of the autophagic vacuoles 17 . Atg5 forms a conjugate with Atg12 and therefore plays a key role in autophagosome formation. P62 is a polyubiquitin-binding protein which contains a LC3-interacting motif and an ubiquitin-binding domain. By linking ubiquitinated substrate with autophagic machinery, p62 is incorporated in completed autophagosomes and degraded in autolysosomes, together with its bound proteins 18 . As shown in Fig. 5c and supplementary Fig. S4,

Stel B promoted autophagic flux in A549 cells.
To elucidate the effect of Stel B on the autophagic process including autophagosome formation, fusion with and degradation in lysosome, we transfected A549 cells with a plasmid stably expressing mRFP (monomeric red fluorescent protein) -GFP (green fluorescent protein) -tagged LC3 (ptfLC3) 19 . At the early stage of autophagy, the ptfLC3 localizes to autophagosomes, developing both GFP (green) and mRFP (red) signals. The GFP protein is acid sensitive and quenched quickly following fusion of autophagosomes with lysosomes, whereas mRFP is relatively stable in the acidic environment of the autolysosome 20 . Before treatment with Stel B, only weak signals of GFP and mRFP protein which represent diffuse LC3 protein were found in the cytoplasm. After treatment with Stel B for 3 h, yellow puncta were observed in the perinuclear region, suggesting the formation of early autophagosomes. Sustained treatment until 17 h resulted in an increased number of autophagic vacuoles, suggesting that autophagosomes gradually developed maturing over time. At the time point of 24 h, the merge picture shows the accumulation of mRFP puncta and the decrease of GFP signal in Stel B treated A549 cells, indicating an increased autophagic flux (Fig. 6).

Stel B inhibited PI3K/Akt/mTOR pathway via reducing p110 expression in A549 cells. PI3K/
Akt/mTOR pathway plays important roles in regulating cell cycle, cell apoptosis and autophagy. To investigate whether the effects of Stel B on A549 cells is related to this pathway, we examined the activity of Stel B on the representative signal proteins in the pathway. As shown in Fig. 7 and supplementary Fig. S4, phosphorylation of Akt, mTOR, p70S6K, as well as GSK-3β , was inhibited dose-dependently after Stel B treatment. Furthermore, phosphorylation of PDK1, which is the upstream activator of Akt was also blocked, implying the direct target of Stel B might be upstream molecules of PDK1. More interestingly, Stel B inhibited the expression of p110, the catalytic subunit of PI3K, in a dose-dependent manner, suggesting that Stel B blocked PI3K/Akt/mTOR pathway   The cells were harvested, and the cell lysates were prepared to be available for western blot analysis for PI3K-p110α , p-PDK1, p-Akt, Akt, p-mTOR, p-p70S6K, p-GSK-3β , p-p38 and p-ERK levels. PI3K-p110α expression was downregulated, and phosphorylation of PDK-1, Akt, mTOR, p70S6K, as well as GSK-3β was inhibited dosedependently after Stel B treatment, while that of p38 and p-ERK was not affected obviously.

Discussion
Marine organisms contain a great number of novel bioactive compounds with potential to be developed as new drugs. To date, fourteen stellettin analogs were isolated from various marine sponges and some of them showed cytotoxic activities [21][22][23][24] . We previously reported Stel B induced apoptosis in neuroblastoma SF295 cells 15 . In this study, we reported for the first time the multifaceted in vitro antitumor activities on A549 cells. Stel B exhibited potent antiproliferative activity on A549 cells with an IC50 as 0.022 μ M. G1 arrest, apoptosis as well as autophagy were induced by Stel B treatment.
Cell proliferation needs cell cycle progression, which is known to be controlled by cyclin-CDK complex and CDK inhibitor proteins. In G1/S checkpoint, cyclin D1 forms a complex with CDK4, and therefore inhibits pRb via phosphorylation, resulting in the release of E2F to promote progression through G1 phase 25 . On the other hand, the activity of CDK4-cyclin D1 complex is negatively controlled by CDK inhibitor proteins including p27 26 . Treatment by Stel B caused reduction in expression of cyclin D1 and phosphorylation of pRb, and enhancement in p27 expression. Therefore, Stel B-induced G1 arrest might be attributed to downregulation of CDK4-cyclin D1 complex and upregulation of p27.
Induction of apoptosis highly affects cell proliferation. Flow cytometry with Annexin V/PI staining suggested that Stel B induced apoptosis in A549 cells, which was supported by the result of DAPI staining assay and increased amount of cleaved PARP. In addition, Stel B significantly promoted ROS generation in A549 cells. It is known that ROS over-production can induce oxidative stress, resulting in apoptosis 27 . Therefore, promotion of ROS generation by Stel B might lead to apoptosis, which could contribute to the antitumor effect of Stel B.
Autophagy is an evolutionarily self-digesting process in which cytoplasmic material is sequestered within cytosolic double-membraned vesicles-autophagosomes, and ended up in the lysosome 28 . In order to investigate the effect of Stel B on autophagy, we utilized various assay methods. MDC staining and TEM showed the formation of autophagosomes. Western blot analysis indicated that the levels of autophagy marker LC3B II/I and Atg5 were increased and the level of p62 was decreased. We also used Tandem mRFP-GFP-LC3 fluorescence assay to confirm the autophagic flux in Stel B-treated cells. As another type of cell death besides of apoptosis, autophagy was frequently reported to be induced by many antitumor agents including taxanes and molecular-targeted agents 29,30 . On the other hand, autophagy was reported to enhance production of ATP, which subsequently binds purinergic receptor P2RX7 in dendritic cells (DC), stimulates the recruitment of DC into the tumor bed, and finally leads to the immunogenic cell death (ICD) of tumor cells 31,32 , suggesting the autophagy induced by Stel B might contribute to the antitumor efficacy.
Finally, we investigated the mechanism which might be involved in the above effects of Stel B. We previously reported that Stel B inhibited phosphorylation of Akt in SF295 cells 15 . Therefore, the effect of Stel B on Akt pathway was examined in A549 cells. As expected, phosphorylation of Akt and the downstream effectors including mTOR, p70S6K and GSK-3β , was inhibited in a dose-dependent manner. Akt is known to increase cyclin D1 through inactivation of GSK-3β and reduce p27 by inhibition of Forkhead family transcription factors and the tumor suppressor tuberin (TSC2) 33 . Therefore, induction of G1 arrest by Stel B might be attributed to the influence on GSK-3β as well as the upstream Akt. It is well known that Akt pathway plays a key role in cell survival, therefore, the apoptosis induced by Stel B might be attributed to the inhibition of Akt phosphorylation. As a downstream effector of Akt, mTOR is known to negatively control autophagy 34 , and mTOR inhibitor rapamycin is well reported as an autophagy inducer 17 . Stel B inhibited phosphorylation of mTOR and p70S6K at a similar concentration to that for autophagy induction in A549 cells, suggesting the autophagy-inducing effect might be attributed to the inhibition of Akt/mTOR pathway.
In order to investigate the target of Stel B in A549 cells, we determined the activity of Stel B on the upstream activators of Akt. As an upstream of Akt and downstream of phosphatidylinositol 3,4,5-trisphosphate (PIP3), PDK1 is phosphorylated by PIP3 and subsequently phosphorylates Akt at Ser308. Phosphatidylinositol 3-kinases (PI3Ks), which contain a catalytic subunit p110 and a regulatory subunit, phosphorylate the 3-hydroxyl group of phosphatidylinositol 4,5-bisphosphate (PIP2) to generate PIP3. Our results showed that Stel B treatment inhibited the phosphorylation of PDK1, and the expression of p110 (Fig. 7). Therefore, the G1 arrest, apoptosis and autophagy inducing effects of Stel B might be attributed to p110 reduction, which leads to inhibition of the downstream effectors like PDK1, Akt, mTOR, as well as GSK-3β .
In conclusion, we isolated Stel B from marine sponge Jaspis stellifera. Stel B treatment induced G1 arrest, apoptosis and autophagy in A549 cells, in which reduction of PI3K-p110 expression and consequent inhibition of PI3K/Akt/mTOR pathway might be closely involved. Since Stel B showed potent activities on human NSCLC A549 cells at low nM doses, and our previous report indicated its very weak cytotoxicity on normal cell lines 15 , it might become a promising drug candidate for NSCLC cancer therapy in the future. However, we realize that it is necessary to exhibit favorable in vivo antitumor activity for stellettin B to become a drug candidate, which remains unclear and will be investigated in our next work.

Cell culture.
Human non-small cell lung cancer A549 cells were obtained from Cell Resource Center, Peking Union Medical College (Beijing, China). The cells were cultured in a humidified incubator with an atmosphere containing 5% CO 2 at 37 °C, and maintained in RPMI1640 medium supplemented with 10% FBS and antibiotics (0.1 μ g/ml of penicillin and 0.1 μ g/ml of streptomycin).
Isolation and structure identification of Stel B. Stel B was isolated from the marine sponge Jaspis stellifera, as described by us previously 15 . Chemical structure of the compound was identified by comparison of the mass and NMR data with those reported previously 35 . The purity of Stel B is 99%.
Cell viability assay. WST assay was used to determine the inhibitory effect of Stel B on the proliferation of A549 cells, as described by us previously 36  Flow cytometric analysis of apoptosis with Annexin V/PI staining. Apoptosis analysis was carried out by detecting phosphatidylserine (PS) externalization using flow cytometer as we reported previously 15 , with a small modification. Briefly, A549 cells were cultured together with 0, 0.02, 0.05, 0.25 and 1 μ M of Stel B for 24 h in 6-well plates. Next day, cells were collected, washed with ice-cold PBS, and resuspended in 50 μ l of binding buffer containing Annexin V-FITC and PI. Then cells were incubated for 15 min in the dark. After dilution, the samples were available for analysis of apoptosis, using flow cytometer FACS Verse (Becton Dickinson, Germany). Data were quantified by using Flow Jo Software (Tristar, CA, USA). DAPI staining. DAPI staining assay was carried out to observe morphological characteristics of apoptotic cells. A549 cell suspension was plated on the coverslips in 6-well plates at a density of 5 × 10 5 cells/well, followed by treatment with 0, 0.02, 0.05, 0.25 and 1 μ M of Stel B for 24 h. ADR, an apoptosis inducer, was used as a positive control. Then, the cells were washed with PBS, fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and then stained with 1 μ g/ml of DAPI solution for 10 min. The fluorescent images of treated samples were obtained using DMI3000B fluorescent microscope (Leica, Germany) with LAS V4.3 software.
Measurement of intracellular ROS generation. ROS measurement was performed by using DCFH-DA, which was de-esterified intracellularly and became highly fluorescent 2′ ,7′ -dichlorofluorescein upon oxidation 37 . A549 cells grown on 6-well plates were treated with 0, 0.02, 0.05, 0.25 and 1 μ M of Stel B for 24 h. The cells were harvested, washed with PBS, and then incubated with 10 μ M of DCFH-DA in the dark at 37 °C for 30 min. The fluorescent signal produced was analyzed by using flow cytometer FACS Verse (BD, Germany). MDC staining. MDC, a specific marker for autophagic vacuoles, was used to examine whether Stel B induced autophagy. A549 cells were grown on the coverslips in 6-well plate, treated with 0, 0.02, 0.05, 0.25 and 1 μ M of Stel B for 24 h. Rapamycin was used as a positive control. The cells were washed with ice-cold PBS, and incubated with 50 μ M of MDC at 37 °C for 30 min. The stained cells were washed, fixed with 4% paraformaldehyde, and analyzed by fluorescence microscope BX51 (Olympus, Japan) with MetaMorph software.
Transmission electron microscopy (TEM). As the most reliable approach for monitoring autophagy, TEM was utilized to confirm autophagy 17 . A549 cells were cultured in 6 cm dishes with 1 μ M of Stel B for 24 h. Cells were collected and fixed. The ultrathin 50 nm sections were cut by use of an ultramicrotome, stained with 2% (w/v) uranyl acetate and lead citrate, then examined with electron microscope Hitachi 600 (Hitachi, Japan).
Analysis of autophagic flux. To analyze autophagic flux, A549 cells were transfected with a tandem fluorescent mRFP-GFP-tagged LC3 plasmid 20 using lipofectamine 2000 according to the manufacturer's instructions. The transfected cells were then treated with 1 μ M of Stel B for 0, 3, 6, 17 and 24 h. The expression of GFP and mRFP was visualized with Olympus FV1000 laser scanning confocal microscope (Olympus, Japan). Images were acquired by using FV10-ASW3.0 software. Autophagic flux was evaluated by the color change of GFP/mRFP at different time points.
Western blot analysis. Western blot analysis was performed as we described previously 38 with a small modification. A549 cells were treated with 0, 0.02, 0.05, 0.25 and 1 μ M of Stel B for 24 h. To prepare the whole cell lysate, the cells were harvested, and lysed with RIPA buffer. For detection of nuclear proteins such as pRb, p27 and cyclin D1, nucleus lysate was prepared using NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific, Rockford, IL, USA). Equivalent amounts of protein were loaded and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to Immobilon-PSQ PVDF