Tuberatolide B Suppresses Cancer Progression by Promoting ROS-Mediated Inhibition of STAT3 Signaling

Tuberatolide B (TTB, C27H34O4) is a diastereomeric meroterpenoid isolated from the Korean marine algae Sargassum macrocarpum. However, the anticancer effects of TTB remain unknown. In this study, we demonstrate that TTB inhibits tumor growth in breast, lung, colon, prostate, and cervical cancer cells. To examine the mechanism by which TTB suppresses cell growth, we determined the effect of TTB on apoptosis, ROS generation, DNA damage, and signal transduction. TTB induced ROS production in MDA-MB-231, A549, and HCT116 cells. Moreover, TTB enhanced DNA damage by inducing γH2AX foci formation and the phosphorylation of DNA damage-related proteins such as Chk2 and H2AX. Furthermore, TTB selectively inhibited STAT3 activation, which resulted in a reduction in cyclin D1, MMP-9, survivin, VEGF, and IL-6. In addition, TTB-induced ROS generation caused STAT3 inhibition, DNA damage, and apoptotic cell death. Therefore, TTB suppresses cancer progression by promoting ROS-mediated inhibition of STAT3 signaling, suggesting that TTB is useful for the treatment of cancer.

Tuberatolide B (TTB, C 27 H 34 O 4 ) is a diastereomeric meroterpenoid isolated from the Korean marine algae Sargassum macrocarpum and acts as a Farnesoid X receptor (FXR) antagonist [26]. However, the effect of TTB on various diseases, including cancer, remains unknown. Here, we report that TTB inhibits cancer growth by promoting ROS-mediated inhibition of STAT3 signaling and inducing DNA damage, thereby suggesting that TTB is useful for the treatment of cancer.

TTB Induces Apoptosis in Cancer Cells
Various cancer cell lines, including breast cancer (MDA-MB-231, MDA-MB-453, and MCF7), lung cancer (A549 and H1299), colon cancer (HCT116, SW620, and CT26), prostate cancer (PC3 and DU145), cervical cancer (HeLa) and non-malignant normal Vero cells were treated with different concentrations (0, 10, 25, 50, and 100 µM) of TTB for 48 h. TTB suppressed cancer cell viability. In addition, TTB did not affect normal monkey kidney epithelial cell viabilities ( Figure 1B). In the live and dead assay, TTB increased the number of dead cells ( Figure 1C). To examine if TTB inhibited cell growth by inducing apoptotic cell death, we investigated the expression of apoptosis-related proteins and the extent of annexin V staining using western blot and flow cytometry, respectively. TTB decreased the expression of Bcl2 and increased the cleavage of caspase-3 and PARP ( Figure 1D). In additions, TTB enhanced the percentage of annexin V-positive apoptotic cells ( Figure 1E). Thus, TTB inhibits cancer cell growth by inducing apoptotic cell death.
Mar. Drugs 2017, 15, 55 2 of 12 pancreatic, and ovarian cancers [19][20][21][22][23]. Accordingly, STAT3 is a potential therapeutic target for cancer treatment, and many STAT3 inhibitors, including synthetic drugs, anti-sense oligonucleotides targeting STAT3, and small molecules derived from natural sources, have been developed to inhibit deregulated STAT3 signaling cascades in cancer [24,25]. Tuberatolide B (TTB, C27H34O4) is a diastereomeric meroterpenoid isolated from the Korean marine algae Sargassum macrocarpum and acts as a Farnesoid X receptor (FXR) antagonist [26]. However, the effect of TTB on various diseases, including cancer, remains unknown. Here, we report that TTB inhibits cancer growth by promoting ROS-mediated inhibition of STAT3 signaling and inducing DNA damage, thereby suggesting that TTB is useful for the treatment of cancer.

TTB Induces Apoptosis in Cancer Cells
Various cancer cell lines, including breast cancer (MDA-MB-231, MDA-MB-453, and MCF7), lung cancer (A549 and H1299), colon cancer (HCT116, SW620, and CT26), prostate cancer (PC3 and DU145), cervical cancer (HeLa) and non-malignant normal Vero cells were treated with different concentrations (0, 10, 25, 50, and 100 μM) of TTB for 48 h. TTB suppressed cancer cell viability. In addition, TTB did not affect normal monkey kidney epithelial cell viabilities ( Figure 1B). In the live and dead assay, TTB increased the number of dead cells ( Figure 1C). To examine if TTB inhibited cell growth by inducing apoptotic cell death, we investigated the expression of apoptosis-related proteins and the extent of annexin V staining using western blot and flow cytometry, respectively. TTB decreased the expression of Bcl2 and increased the cleavage of caspase-3 and PARP ( Figure 1D). In additions, TTB enhanced the percentage of annexin V-positive apoptotic cells ( Figure 1E). Thus, TTB inhibits cancer cell growth by inducing apoptotic cell death.

TTB Increases ROS Generation in Cancer Cells
The regulation of intracellular ROS generation plays a role in many cellular functions, such as cell proliferation and apoptosis, which are critical processes in tumor development. To investigate the effect of TTB on ROS production, we assessed ROS generation using flow cytometry. When compared to the control, TTB increased ROS generation by approximately 67%, 36%, and 52% in MDA-MB-231, A549, and HCT116 cells, respectively ( Figure 2A). Moreover, the well-known ROS scavenger NAC suppressed TTB-mediated ROS production in cancer cells ( Figure 2B). Therefore, TTB induces ROS generation in many types of cancer cells.

TTB Induces DNA Damage in Cancer Cells
DNA damage-inducing drugs that cause the apoptotic cell death of cancer cells may be a viable cancer treatment [27,28]. Therefore, we examined effect of TTB on DNA damage. γ-H2AX staining is a well-known marker of oxidative-related DNA damage [29,30]. TTB increased co-staining of DAPI and γH2AX foci in MDA-MB-231 cells ( Figure 3A). In additions, TTB induced the phosphorylation of Chk2 and H2AX in MDA-MB-231, A549 and HCT-116 cells ( Figure 3B). Moreover TTB enhanced DNA fragmentation in MDA-MB-231 cells when compared with the control ( Figure 3C). Thus, TTB increases DNA damage in cancer cells.

TTB Increases ROS Generation in Cancer Cells
The regulation of intracellular ROS generation plays a role in many cellular functions, such as cell proliferation and apoptosis, which are critical processes in tumor development. To investigate the effect of TTB on ROS production, we assessed ROS generation using flow cytometry. When compared to the control, TTB increased ROS generation by approximately 67%, 36%, and 52% in MDA-MB-231, A549, and HCT116 cells, respectively ( Figure 2A). Moreover, the well-known ROS scavenger NAC suppressed TTB-mediated ROS production in cancer cells ( Figure 2B). Therefore, TTB induces ROS generation in many types of cancer cells.

TTB Induces DNA Damage in Cancer Cells
DNA damage-inducing drugs that cause the apoptotic cell death of cancer cells may be a viable cancer treatment [27,28]. Therefore, we examined effect of TTB on DNA damage. γ-H2AX staining is a well-known marker of oxidative-related DNA damage [29,30]. TTB increased co-staining of DAPI and γH2AX foci in MDA-MB-231 cells ( Figure 3A). In additions, TTB induced the phosphorylation of Chk2 and H2AX in MDA-MB-231, A549 and HCT-116 cells ( Figure 3B). Moreover TTB enhanced DNA fragmentation in MDA-MB-231 cells when compared with the control ( Figure 3C). Thus, TTB increases DNA damage in cancer cells.

TTB Selectively Inhibits the STAT3 Signaling Pathway in Cancer Cells
To further elucidate the anticancer effects of TTB on cancer cells, we identified which intracellular signaling pathways were involved. Cells were treated with 100 μM TTB for 15 min and then subjected to western blotting. STAT3 phosphorylation was selectively and strongly suppressed by TTB. The phosphorylation of EGFR (Y992, Y1068, and Y1173), AKT, ERK, JNK, and p38 remained unchanged after TTB treatment ( Figure 4A). Next, to confirm the TTB-mediated inhibition of the STAT3 pathway, we examined the transcriptional activation of STAT3 using a luciferase assay. As shown in Figure 4B, TTB reduced STAT3 transcriptional activity when compared with the control. Next, we assessed the effect of TTB on the expression of STAT3-target genes. TTB decreased the protein expression levels of many STAT3-target genes, including cyclin D1, MMP-9 and survivin ( Figure 4C). TTB also reduced the secreted protein levels of VEGF, MMP-9, and IL-6 in MDA-MB-231 cells ( Figure 4D). Thus, our data suggest that TTB selectively suppresses STAT3 activity and STAT3dependent gene expression.

TTB-Induced ROS Generation Causes STAT3 Inhibition and Apoptosis
ROS generation inhibits STAT3 signaling [14,31]; thus, we examined STAT3 expression in cells after co-treatment with TTB and NAC. As shown in Figure 5A, the TTB-mediated reduction in phosphorylated STAT3 was restored after treatment with NAC. Furthermore, co-treatment with TTB and NAC increased STAT3-dependent gene expression (VEGF and MMP-9) when compared with the TTB treatment, suggesting that the inhibition of STAT3 pathway activation by TTB was at least partially attributed to ROS generation in MDA-MB-231 cells ( Figure 5B). ROS generation induces DNA damage [32]; thus, we evaluated the role of ROS in TTB-induced DNA damage by treating cells with NAC. Indeed, TTB-induced γH2AX foci were abolished in the presence this ROS inhibitor ( Figure 5C). In addition, NAC reduced TTB-mediated apoptotic cell death ( Figure 5D). Therefore, TTB-induced DNA damage, apoptosis and reduced STAT3 activity occurs via ROS generation in cancer cells.

TTB Selectively Inhibits the STAT3 Signaling Pathway in Cancer Cells
To further elucidate the anticancer effects of TTB on cancer cells, we identified which intracellular signaling pathways were involved. Cells were treated with 100 µM TTB for 15 min and then subjected to western blotting. STAT3 phosphorylation was selectively and strongly suppressed by TTB. The phosphorylation of EGFR (Y992, Y1068, and Y1173), AKT, ERK, JNK, and p38 remained unchanged after TTB treatment ( Figure 4A). Next, to confirm the TTB-mediated inhibition of the STAT3 pathway, we examined the transcriptional activation of STAT3 using a luciferase assay. As shown in Figure 4B, TTB reduced STAT3 transcriptional activity when compared with the control. Next, we assessed the effect of TTB on the expression of STAT3-target genes. TTB decreased the protein expression levels of many STAT3-target genes, including cyclin D1, MMP-9 and survivin ( Figure 4C). TTB also reduced the secreted protein levels of VEGF, MMP-9, and IL-6 in MDA-MB-231 cells ( Figure 4D). Thus, our data suggest that TTB selectively suppresses STAT3 activity and STAT3-dependent gene expression.

TTB-Induced ROS Generation Causes STAT3 Inhibition and Apoptosis
ROS generation inhibits STAT3 signaling [14,31]; thus, we examined STAT3 expression in cells after co-treatment with TTB and NAC. As shown in Figure 5A, the TTB-mediated reduction in phosphorylated STAT3 was restored after treatment with NAC. Furthermore, co-treatment with TTB and NAC increased STAT3-dependent gene expression (VEGF and MMP-9) when compared with the TTB treatment, suggesting that the inhibition of STAT3 pathway activation by TTB was at least partially attributed to ROS generation in MDA-MB-231 cells ( Figure 5B). ROS generation induces DNA damage [32]; thus, we evaluated the role of ROS in TTB-induced DNA damage by treating cells with NAC. Indeed, TTB-induced γH2AX foci were abolished in the presence this ROS inhibitor ( Figure 5C). In addition, NAC reduced TTB-mediated apoptotic cell death ( Figure 5D). Therefore, TTB-induced DNA damage, apoptosis and reduced STAT3 activity occurs via ROS generation in cancer cells.    Cells were stained with anti-γH2AX antibody and DAPI was used for nuclear staining. Images were obtained with using Olympys FV10i Self Contained Confocal Laser System. The object was 20× and scale bar indicates 10 µm. * p < 0.05; (D) HCT116 cells were pretreated with NAC for 1 h and then exposed to TTB (100 µM) for 48 h. Cells were stained with Annexin V and 7AAD at room temperature in the dark. Experiments were performed in triplicate. Bar indicate means and standard deviations. * p < 0.05; (E) A schematic representation of the mechanisms for TTB suppressed cancer cell growth.

Discussion
Cancer still remains a deadly disease and has a high incidence and death rate worldwide [25]. Unlike normal cells, cancer cells have some characteristics, such as sustained proliferative signaling, evaded growth suppressors, activated invasion, and metastasis, that enable replicative immortality, induce angiogenesis, and resist cell death [33]. Therefore, targeted cancer therapeutic agents are developed for cancer patients for a long time [34]. Especially because constitutive STAT3 activation is associated with poor prognosis in cancer patients, STAT3 has been investigated as a cancer therapeutic target [35,36]. Therefore, STAT3 specific inhibitor may be useful cancer treatment and many STAT3 inhibitors are in the process of being tested in clinical trials [36]. Phosphorylated tyrosine-705 STAT3 (Y-705) is required for STAT3 dimerization and nuclear translocation. Dimeric STAT3 bind to specific DNA response elements in the promoters of target genes [37]. In our study, TTB selectively suppressed STAT3 phosphorylation, transcriptional activity and expression of target genes such as Cyclin D1, MMP-9, Survivin, and IL-6.
Our data indicate that TTB induction of ROS level is a key modulate to enhance the apoptosis in various cancer cells. In addition, NAC rescued TTB-mediated apoptosis. ROS can be generated from exogenous sources, such as chemical, pharmaceutical, and endogenous sources, including mitochondria, activation of inflammatory cells, and peroxisomes [38,39]. Importantly, numerous studies have shown that many cancer chemotherapeutic drugs have anticancer effects by inducing ROS-mediated apoptosis. For example, the classic anticancer drugs adriamycin and cisplatin induces excessive levels of ROS, resulting in DNA damage and apoptotic cell death [39]. Moreover, a number of natural compounds, such as tocopheryl succinate (a vitamin E analog), c-phycocyanin (a major phycobiliprotein from blue-green algae), and β-phenylethyl isothiocyanate (PEITC), are reported to induce ROS production and kill cancer cells [7,40,41]. Thus, ROS is crucial for inducing cell death in cancer cells. Moreover, ROS are inducing DNA damage, resulting in single-or double-strand breakage, DNA cross-linking, and base modification, and these events can result in cell death [42]. DNA double-strand breaks (DSBs) induce H2AX phosphorylation on serime 139, then called gamma-H2AX (γ-H2AX) and γ-H2AX foci formation at DSB sites occurs rapidly [43,44]. Furthermore serin/threonine kinase Chk2 is a major regulator of the DNA damage response [45] and phosphorylated Chk2 is essential for H2AX phosphorylation [46]. In our study, TTB induced γ-H2AX foci formation and increased phosphorylation of Chk2 and H2AX levels. Moreover, TTB enhanced DNA fragmentation. In additions, the ROS scavenger NAC inhibited TTB-mediated γ-H2AX foci formation and apoptotic cell death in cancer cells.

Extraction and Isolation of TTB
The brown alga Sargassum macrocarpum was collected from along the coast of Jeju Island, Korea. The sample was washed thrice with tap water to remove salt, sand, and epiphytes attached to its surface, followed by careful rinsing with fresh water and freezing in a medical refrigerator at −20 • C. Thereafter, the frozen sample was lyophilized and homogenized with a grinder prior to extraction. All chemicals and reagents used were of analytical quality and sourced from trusted commercial sources (grade ≥ 95%). The dried S. macrocarpum powder was extracted thrice with 80% aqueous methanol at the room temperature. The liquid layer was obtained via filtration, and the filtrate was concentrated by using an evaporator under reduced pressure. The extract was suspended in water, and the aqueous layer was partitioned with chloroform. Then, the chloroform fraction was fractionated by silica column chromatography with stepwise elution of chloroform-methanol mixture (50:1→1:1) to separate active fractions in chloroform extract. A combined active fraction was further subjected to a Sephadex LH-20 column saturated with 100% methanol, and then purified by reversed phase high performance liquid chromatography (HPLC) using a Waters HPLC system (Alliance 2690; Waters Corp., Milford, MA, USA) equipped with a Waters 996 photodiode array detector and C18 column (J'sphere ODS-H80, 250 × 4.6 mm, 4 µm; YMC Co., Kyoto, Japan) by stepwise elution with methanol-water gradient (UV range, 220 nm; flow rate, 1 mL/min). Finally, the purified compound was identified by comparing its 1H and 13C NMR data with literature [26]. The chemical structure of tuberatolide B is indicated in Figure 1. The compound was dissolved in dimethylsulfoxide (DMSO) and employed in experiments in which the final concentration of DMSO in culture medium was adjusted to <0.01%.

Cell Viability and Apoptotic Analysis
Various cancer cells were seeded on 96-well plates and treated with TTB for 48 h. Cell viability was determined using the MTT assay (Sigma-Aldrich, St. Louis, MO, USA). Absorbance was read at 570 nm on the ELISA reader (Molecular Devices, Palo Alto, CA, USA). Cells were treated with TTB for 48 h and then resuspended in binding buffer. After cells were stained with Annexin V-FITC (BD Bioscience, San Jose, CA, USA) in the dark at room temperature for 15 min, Annexin V-stained cells were incubated with 7AAD in the dark at room temperature for 15 min. Annexin V-and 7AAD-positive cells were detected by FACSCalibur flow cytometry (BD Bioscience, San Jose, CA, USA). Live and dead assay was performed with the live and dead cell assay kit (Abcam, Cambridge, UK) according to the manufacturer's instruction.

ROS Measurement and DNA Fragmentation Assay
Cells were seeded in 6-well plates and treated with TTB and H2DCFDA for 1 h at 37 • C. After harvested, the data was analyzed by FACSCalibur flow cytometry measuring by the FL1 channel. For the inhibition of ROS generation, cells were pretreated with N-acetyl-L-cysteine (NAC, 2.5 mM, Sigma-Aldrich, St. Louis, MO, USA) for 1 h before TTB and H2DCFDA co-treatment. The data was analyzed by FACSCalibur flow cytometry. For DNA fragmentation, cells were seed in 100 mm dishes and treated with TTB for 24 h. After harvest, cells were lyzed with DNA isolation buffer (0.1 M NaCl, 0.01M EDTA, 0.3M Tris-HCl (pH 7.5) and 0.2 M sucrose) and DNA gel electrophoresis was performed.

Luciferase Assay and ELISA
Cells were seeded in 24-well plates and p4xM67-TK-luc plasmid (Addgene, Cambridge, MA, USA) was transfected in MDA-MB-231 cells by using Lipofectamine reagent (Invitrogen, Carlsbad, CA, USA). Cells were treated with TTB for 6 h, and then luciferase assay was performed by using dual-luciferase reporter assay kits (Promega, Madison, WI, USA) according to the manufacturer's instructions. All transfections included the RLTK-Luc (kindly provide by Sang Hoon Kim) for transfection efficiency. For ELISA assay, cells were seeded in 6-well plates and treated with TTB. After 24 h, supernatants were harvested and secreted protein levels of VEGF, MMP-9, and IL-6 were performed with human VEGF and MMP-9 ELISA kits (R&D Systems, Minneapolis, MN, USA) and human IL-6 ELISA kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer's instructions.

Statistics
All the data were performed in triplicate, and shown as means and standard deviations. p-values less than 0.05 in the two-tailed Student's t-test were considered significant.

Conclusions
In conclusion, we provide evidence for the first time that anticancer effect of TTB on diverse cancer cells result from the induction of ROS-mediated apoptosis by inhibiting of STAT3 phosphorylation and enhancing of DNA damage. Therefore, TTB might be an effective and useful chemotherapy agent against cancer.