Fucoidan from marine brown algae attenuates pancreatic cancer progression by regulating p53 – NFκB crosstalk

Phytochemistry 167 (2019) 112078

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Fucoidan from marine brown algae attenuates pancreatic cancer progression by regulating p53 – NFκB crosstalk

T

Caroline R. Delmaa,b,∗,1, Somasundaram Thirugnanasambandana, Guru Prasad Srinivasana, Nune Raviprakashc, Sunil K. Mannac, Mohan Natarajanb, Natarajan Aravindand a

Centre of Advanced Study in Marine Biology, Faculty of Marine Sciences, Annamalai University, Parangipettai, TN, India Department of Pathology, University of Texas Health Sciences Center at San Antonio, TX, USA c Laboratory of Immunology, Centre for DNA Fingerprinting and Diagnostics, Hyderabad, AP, India d Department of Radiation Oncology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA b

A R T I C LE I N FO

A B S T R A C T

Keywords: Turbinaria conoides Sargassaceae Pancreatic cancer Fucoidan Apoptosis NFκB p53

Poor pancreatic cancer (PC) prognosis has been attributed to its resistance to apoptosis and propensity for early systemic dissemination. Existing therapeutic strategies are often circumvented by the molecular crosstalk between cell-signalling pathways. p53 is mutated in more than 50% of PC and NFκB is constitutively activated in therapy-resistant residual disease; these mutations and activations account for the avoidance of cell death and metastasis. Recently, we demonstrated the anti-PC potential of fucoidan extract from marine brown alga, Turbinaria conoides (J. Agardh) Kützing (Sargassaceae). In this study, we aimed to characterize the active fractions of fucoidan extract to identify their select anti-PC efficacy, and to define the mechanism(s) involved. Five fractions of fucoidan isolated by ion exchange chromatography were tested for their potential in genetically diverse human PC cell lines. All fractions exerted significant dose-dependent and time-dependent regulation of cell survival. Fucoidans induced apoptosis, activated caspase −3, −8 and −9, and cleaved Poly ADP ribose polymerase (PARP). Pathway-specific transcriptional analysis recognized inhibition of 57 and 38 nuclear factor κB (NFκB) pathway molecules with fucoidan-F5 in MiaPaCa-2 and Panc-1 cells, respectively. In addition, fucoidan-F5 inhibited both the constitutive and Tumor necrosis factor-α (TNFα)-mediated NFκB DNA-binding activity in PC cells. Upregulation of cytoplasmic IκB levels and significant reduction of NFκB-dependent luciferase activity further substantiate the inhibitory potential of seaweed fucoidans on NFκB. Moreover, fucoidan(s) treatment increased cellular p53 in PC cells and reverted NFκB forced-expression-related p53 reduction. The results suggest that fucoidan regulates PC progression and that fucoidans may target p53–NFκB crosstalk and dictate apoptosis in PC cells.

1. Introduction Pancreatic cancer (PC) is one of the most aggressive cancers, and is the fourth leading cause of cancer-related deaths in United States, with an estimated 5-year survival rate of less than 8% (Society, 2017). The mortality rate has not changed over the past three decades; the poor prognosis can be attributed to the presence of distant metastasis at the time of diagnosis in about 52% of patients, who then have a 5-year overall survival rate of 3% (Society, 2017). More than 90% of cases are resistant to existing chemotherapies (Morton et al., 2010; Mulcahy et al., 2005). Currently, the nucleoside analog gemcitabine is the drug of choice for PC treatment, but gemcitabine appears to have a palliative

role, rather than increasing the survival rate of patients with PC (Khanbolooki et al., 2006). Extensive research is being performed towards the development of targeted therapies to treat PC. In addition, 968 clinical trials are ongoing (www.clinicaltrials.gov, assessed on February 2018). However, targeted therapies are circumvented by the molecular crosstalk between cell signalling pathways that eventually results in cancer progression and metastasis (Aravindan et al., 2013a). Thus, there is a need to develop more potent regimens, including identification of novel anti-cancer drugs with fewer adverse effects from natural resources. In recent years, the untapped marine environment has grabbed the attention of scientists worldwide who strive to develop more effective chemotherapeutic agents. Marine compounds are less

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Corresponding author. Department of Pathology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX, 78229, USA. E-mail address: [email protected] (C.R. Delma). 1 Present address of the corresponding author https://doi.org/10.1016/j.phytochem.2019.112078 Received 1 February 2019; Received in revised form 29 July 2019; Accepted 30 July 2019 0031-9422/ © 2019 Elsevier Ltd. All rights reserved.

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water and lyophilized. Downstream analysis for the total sugar content in the fractions revealed 42.12%, 27.56%, 43.44%, 66.23%, and 51.23% in fucoidan fractions F1, F2, F3, F4, and F5 respectively. Moreover, we observed a measurable content of uronic acid in all fucoidan fractions (Table 1). The uronic acid content was minimal in fraction F5 compared with the other fractions. In addition, we observed a strong sulfate content in the extracted fucoidan fractions (Table 1). Interestingly, we found very high levels of sulfate content in fraction F5 compared with the other fractions. Fraction F2 had very low sulfate content.

toxic and have enormous biomedical potential as prospective candidates for the treatment of PC (Aravindan et al., 2013a, 2015a, 2015b, 2017; Geisen et al., 2015). Fucoidans, which are sulfated polysaccharides from marine brown algae, have been reported to possess antagonistic activities (Atashrazm et al., 2015; Fitton et al., 2015a) against several cancer types, including cancers of the breast (Xue et al., 2017), lung (Hsu et al., 2017), prostate (Boo et al., 2013), colon (Han et al., 2015; Ikeguchi et al., 2011), melanoma (Dithmer et al., 2017), and lymphoma (Yang et al., 2015). However, our understanding of the beneficial effect of fucoidans on PC regression is at the very early stage. Our recent investigations indicated the multi-dimensional anti-PC potential of the fucoidan extract from marine seaweed, Turbinaria conoides (J. Agardh) Kützing and it has been found to be non-toxic to normal cells (Delma et al., 2015). In the present study, we characterized the fractions of the fucoidan extract and investigated the fucoidan fractions’ targeted molecular events and translation of functional signalling in orchestrated anti-PC potential. The nuclear transcription factor kappa B (NFκB) has been implicated in the evolution of many cancers. NFκB is rendered inactive by its sequestration in the cytoplasm by the inhibitory protein, IκB. Once released from its inhibitor, NFκB is translocated to the nucleus, where it binds to specific promoter sites to regulate the transcription of many genes associated with oncogenesis and tumor progression (Bonizzi and Karin, 2004; Jobin and Sartor, 2000; Nakanishi and Toi, 2005). NFκB is one of the key factors in conferring apoptotic resistance to PC cells (Qian et al., 2017). It has been reported to be constitutively activated in PC cells, but not in normal pancreatic tissues (Wang et al., 1999). Various studies have shown that inhibition of NFκB antagonizes the survival of PC cells and sensitizes them to chemotherapy (Du et al., 2013; Liu et al., 2011). Constitutive activation of NFκB inhibits apoptosis by inducing anti-apoptotic genes and/or suppressing pro-apoptotic genes. Several chemotherapeutic agents also activate NFκB, which in turn causes chemoresistance (Nakanishi and Toi, 2005). While NFκB is known to inhibit apoptosis, the transcription factor p53, which is a well-known tumor suppressor gene, is an inducer of apoptosis. Crosstalk exists between p53 and NFκB signalling. p53 and NFκB antagonize each other, as they both employ the same histone acetyl transferase enzymes (p300/CBP) for their transcriptional regulation (Ravi et al., 1998; Webster and Perkins, 1999). p53 activation negatively regulates the expression of RelA (p65), the subunit of NFκB and NFκB activity, and vice versa. This inverse relationship paves the way for devising new therapeutic strategies to combat cancer progression. It is better to use dietary agents to sensitize cancer cells to chemotherapy by inhibiting NFκB, as these agents are non-toxic to other non-cancerous tissues. Fucoidans, which are used as a dietary supplement and in traditional medicine, may be potential candidates against PC. Although there are several reports on the apoptosis-inducing activity of fucoidans, their impact on the mechanisms driving apoptosis, including p53-NFκB crosstalk in PC or in any tumor system, has not been investigated. In the present study, we demonstrated the anti-PC potential of select fucoidan fractions from the marine brown alga, Turbinaria conoides. The results indicate that fucoidans negatively regulate NFκB signalling and mediate apoptosis in PC cell lines through p53 activation.

2.2. Fucoidans inhibit PC cell proliferation in vitro The effect of fucoidans on the survival of PC cells was determined by MTT assay. To assess dose-dependent anti-proliferative potential, a range of concentrations (3.125, 6.25, 12.5, 25, 50 μg/ml) of each fucoidan fractions was used. All five fucoidan fractions exerted a marked and dose-dependent inhibition of PC cell survival (Fig. 2 A&B), although the degree of inhibition varied between fractions and cell lines. The proliferation inhibition was marginal with the 3.125μg/ml dose, but we observed a consistent and significant dose-dependent inhibition of cell survival, with maximal inhibition at the 50μg/ml dose in both cell lines investigated. We observed a heightened immediate early effect (24 h post-treatment) of the drugs in Panc-1 cells compared with MiaPaCA-2 cells (Fig. 2 A&B). To examine whether the fucoidan fraction-induced inhibition of cell survival is a transient or definite process, cells treated with doses of fucoidan fractions were examined at 72 h after treatment. Interestingly, these fractions not only maintained their PC cell proliferation inhibition capabilities, but also showed increased inhibition over an extended period (Fig. 2 C&D). Notably, we observed a complete (> 80%) inhibition of cell proliferation with fraction F5 in MiaPaCa-2 cells. Although fraction F2 has slightly better inhibitory potential in Panc-1 cells with certain doses, fraction F5 showed consistent overall dominance in PC cell proliferation inhibition (Fig. 2 C& D). Conversely, fraction F1 was the least effective in survival inhibition in both cell lines, and was excluded from the downstream experimental approach.

2.3. Fucoidans endorse apoptosis in PC cells To further define that fucoidan fractions exert anti-PC potential by orchestrating programmed cell death, we investigated whether treatment with fucoidan fractions induces apoptosis. One of the most important features of apoptosis is the activation of highly specialized family of cysteinyl-aspartate proteases called caspases. To obtain insight into the apoptosis-inducing potential of fucoidans, we investigated the role of caspases in treated PC cells. Compared with the untreated controls, immunoblotting analysis revealed a profound increase in the cleaved caspase 3, 8, and 9 in MiaPaCa-2 and Panc-1 cells treated with fucoidan fractions. Despite the cell-line-dependent and fraction-dependent changes observed in the cleavage of these apoptotic machinery proteins, fucoidan fraction 5 produced unparalleled activation of cleaved caspases (Fig. 3). PARP-1, the key cellular substrate of caspases, and the cleavage of PARP-1 by caspases is one of the hallmarks of apoptosis (Kaufmann et al., 1993; Tewari et al., 1995). We observed a significant cleavage of PARP with the fucoidan treatment in both PC cell lines investigated (Fig. 3). More importantly, we observed a significant increase in PARP cleavage in F5-treated cells compared with cells treated with the other fractions. The induction of the apoptosis machinery architects and apoptosis with the fucoidan fractions directly indicate the potential of fucoidans orchestrating caspases-dependent pancreatic cancer cell death.

2. Results 2.1. Fucoidan fraction separation and their physiognomies Our earlier study indicated the anti-pancreatic cancer potential of crude fucoidan extract from marine brown alga, T. conoides. It is crucial to characterize the components of the crude extract and identify their efficacy in PC settings. In this regard, our anion exchange chromatography analysis indicated five peaks, including two dominant peaks. Utilizing the hot water extraction method on a DEAE cellulose-52 column, we isolated all five fucoidan fractions: F1, F2, F3, F4, and F5 (Fig. 1). The extracted fucoidan fractions were dialyzed against distilled 2

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Fig. 1. Elution profile of fucoidan extract from Turbinaria conoides. Fucoidan extract was separated on a DEAE cellulose 52 column by anion exchange chromatography. The fractions were eluted with a linear gradient of 0–3.0M NaCl in sodium acetate buffer (pH 5.0) and were analyzed for carbohydrate content. The chromatography analysis indicated five peaks that were named fucoidan fractions F1, F2, F3, F4, and F5.

2.5. Fucoidan inhibits NFκB activation and favors apoptosis through p53 expression

Table 1 Chemical composition of fucoidan fractions from T. conoides. Fraction

Total Sugar (%)

Uronic acid (%)

Sulfate (%)

F1 F2 F3 F4 F5

42.13 27.56 43.44 66.23 51.23

17.66 18.30 15.38 13.56 6.61

10.11 31.59 49.59 63.46 73.78

Several reports have suggested the constitutive activation of NFκB in PC, which is often correlated with the chemoresistance of PC cells to apoptosis (Wang et al., 1999). NFκB activation acts as an inhibitor of apoptosis by regulating a variety of anti-apoptotic genes (Wang et al., 1998). In contrast, the tumor suppressor p53 plays a critical role in inducing cell cycle arrest, apoptosis, or senescence, and is frequently mutated in 50–70% of human PC (Scarpa et al., 1993). An inverse correlation between p53 and NFκB has been reported in several cancers (Webster and Perkins, 1999). To substantiate our finding that fucoidan F5 inhibits the NFκB signalling pathway, we examined the effect of fucoidan fraction F5 on the DNA-binding activity of NFκB. We further defined the benefit of fucoidan fraction F5 in inhibiting activated NFκB, in this case with exogenous TNFα. MiaPaCa-2 cells were treated with 5 and 10 μg/ml of F5 and were examined for alterations in NFκB DNA binding activity., EMSA analysis revealed a complete inhibition of the DNA binding activity in fucoidan-treated cells compared with the untreated control (Fig. 5A&B). Next, we investigated the benefit of fucoidan in inhibiting TNFα-induced NFκB DNA binding activity. Cells were treated with exogenous TNFα (1 ng/ml) for 1 h and were then treated with various concentrations (1, 2, 5, 10 μg/ml) of fucoidan fractions. Compared with the untreated controls, TNFα significantly activated the DNA binding activity of NFκB in PC cells (Fig. 5A&B). Treatment with the fucoidan fraction exerted a significant dose-dependent inhibition of TNFα-induced NFκB DNA binding activity in these cells (Fig. 5A&B). Furthermore, luciferase reporter assay results revealed a significant inhibition of NFκB-dependent luciferase gene activity in MiaPaCa-2 cells treated with fucoidan F5 (Fig. 5C). In addition, immunoblot analysis from the cytoplasmic extracts of PC cells treated with fucoidan in the presence of TNFα revealed that the levels of IκBα were increased in cells treated with fraction F5 compared with cells treated with TNFα alone, suggesting the inhibition of IκBα degradation (Fig. 5D). This stabilization of IκBα with the fucoidan remained consistent over time, suggesting the maintenance of IκBα stabilization with fucoidan F5 (Fig. 5D). However, we did not see any considerable difference in the cytoplasmic levels of p65 compared with the TNFα controls. These results, in conjunction with our observations of fucoidan F5-inflicted, dose-dependent reductions in NFκB DNA binding activity and its transcriptional function, affirm the benefit of F5 in this setting. Further, our findings indicate that p65 is sequestered in the cytoplasm, and only its DNA binding activity and function at the nuclear level are attenuated by the fucoidan fraction F5 (Fig. 5D). Furthermore, our observations revealed robust and significant (P < 0.001) induction of p53 expression in PC cells treated with

Fucoidan fractions were separated by anion exchange chromatography. The chemical characteristics of individual fractions (F1, F2, F3, F4 and F5), total sugar (TS), uronic acid, and sulfate content were determined.

2.4. Fucoidan fraction F5 targets the NFκB signalling pathway transcription in human PC cells To define the efficacy of fucoidan fraction F5 in targeting disease progression signalling, we investigated the alterations in mRNA levels for 84 well-characterized NFκB signalling pathway (up/down stream) molecules (Table S1) in genetically diverse human PC cells. QPCR profiling revealed unique amplification signatures across cell lines. Profile-to-profile expression distinctions were normalized with in-house controls (HPRT-1, GAPDH, and/or β-actin). Overall, fraction F5 resulted in the inhibition of 66, 35, 25, and 48 NFκB signalling pathway molecules in MiaPaCa-2, Panc-1, Panc-3.27, and BX-PC3 cells, respectively. Interestingly, cells→genes traverse analysis identified cell-lineindependent inhibition of three genes, BCl10, IFNA1, and TNFRSF10B, across all four cell lines (Fig. 4). Twenty-one genes, BCL2L1, BCL3, BIRC2, BIRC5, CCL2, CHUK, F2R, FADD, HMOX1, IL12A, IRAK1, JUN, LTA, MAP3K1, NLRP12, NOD1, PPM1A, TLR4, TLR9, TMED4, and TRADD, showed cell-line-independent inhibition in at least three cell lines (Fig. 4). In addition, a set of 42 NFκB signalling pathway molecules, including ATF-1, BCL 2, CASP8, CD40, EDARADD, EDG2, EGR1, ELK1, GJA1, HTR2B, ICAM1, IFNA2, IFNG, IKBKE, IKBKG, IL10, IL12B, IL1A, IL6, IL8, IRAK2, LTBR, MALT1, MMP7, MYD88, NFKB1, NFKB2, NFKBIA, RAF1, REL, RELB, RHOA, SELL, STAT1, TBK1, TICAM2, TLR1, TLR2, TLR7, TNF, TNFRSF10A, and TNFSF10, showed cell-line-independent inhibition in two cell lines (Fig. 4). Distinctively, a small subset of genes showed cell-line-dependent inhibition in Panc-1 (IKBKB), Panc-3.27 (Casp1, TLR8, AKT-1), BxPC-3 (TLR3, CFLAR, RIPK1, Fos), and MiaPaCa-2 (IL-1b, IL1-R1, Csf2, Ticam1, TNAIP3, TNFRSF1A, TNFSF14) cells. These results highlight the inhibition profile of NFκB signalling pathway transcripts in PC cells treated with fucoidan fraction F5. Further, these data identify the cell-line-independent and cell-line-dependent inhibition in NFκB signalling transcriptional responses in PC cells with F5 treatment. 3

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Fig. 2. Anti-proliferative effect of fucoidan fractions. The fractions exhibited an overall dose-dependent anti-proliferative activity in both MiaPaCa-2 and Panc-1 cells. The survival rate of Panc-1 and MiaPaCa-2 cells against fucoidan fractions (F1 through F5) was determined at 24 h (A&B) and 72 h (C&D). All five fractions exerted cell-specific inhibitory effects on the proliferation of pancreatic cancer cells. Data are mean ± standard deviation in triplicate. Significance was determined by ANOVA with Bonferroni's post-hoc correction.

fucoidans (Fig. 6A). To define whether fucoidan intervenes with NFκB – p53 cross-talk that could orchestrate the mediated PC cell apoptosis, we investigated the effect of fucoidan F5 in reverting the NFκB associated regulation of p53. Compared to the untreated controls, ectopic expression of NFκB resulted in reduced levels of p53 in MiaPaCa-2 cells (Fig. 6B). Conversely, treating the NFκB-expressing cells with fucoidan F5 robustly increased p53 levels, demonstrating that fucoidan F5 can target NFκB in PC cells and thereby orchestrate the activation of p53 and promote apoptosis.

crude extract in PC settings (Delma et al., 2015). Five fractions were obtained and their chemical compositions were analyzed. Overall, fraction F2 had the least carbohydrate content, whereas fractions F4 and F5 had relatively higher carbohydrate content. This result is in accordance with the previous studies of seaweed polysaccharides (Qi et al., 2005; Wang et al., 2008). Similarly, the sulfate and uronic acid content were higher in fraction F4 and F5, and lowest in fraction F1. Based on these observations, F1 had the lowest negative charge and F5 had the highest negative charge, which explains the order of elution from the anion exchange column. Several studies have substantiated the inhibitory effect of fucoidans on the proliferation of various cancer cell lines (Patel, 2012). However, the potential of these carbohydrate moieties on pancreatic cancer have not been reported so far. In the present study, purified fractions of fucoidan were found to markedly attenuate the proliferation of PC cell lines in a dose-dependent and time-dependent manner. Fraction F5 profoundly inhibited the proliferation of MiaPaCa-2 after 72 h. However, F1 exhibited the least activity in both cell lines. It has been reported that the bioactivities of sulfated polysaccharides depend on various structural parameters, such as degree and position of sulfation, molecular weight, and type of sugar, of which the main factor that determines the bioactivity is the sulfate content (Aisa et al., 2005; Ale et al., 2011; Duarte et al., 2001). Fraction F5 has the highest sulfate content of all of the fractions. This content accounts for the differences

3. Discussion The present study demonstrates the inhibitory potential of fucoidan isolated from the brown alga Turbinaria conoides, and its role in NFκBmediated gene regulation. Fucoidans have been traditionally used as a dietary supplements in many Asian countries. They possess diverse properties in terms of biomedical potential, such as antioxidant, antiinflammatory, anti-cancer, anti-metastatic, and anti-angiogenic capabilities (Koyanagi et al., 2003; Tang et al., 2006). Because of their orally consumable and non-toxic nature, fucoidans have proven to be excellent candidates for cancer treatment regimens. In the present study, fucoidans were purified from the crude polysaccharide extract of T. conoides by anion exchange chromatography. Previously, we demonstrated the beneficial anti-cancer potential of this 4

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Fig. 3. Effect of fucoidan on the apoptotic pathway. MiaPaCa-2 and Panc-1 pancreatic cancer cells were treated with 5 μg/ml of the fucoidan fractions for 24 h. The protein levels of apoptotic markers, Caspase-3, -8 and -9, and cleaved PARP were assessed by western blotting. The blots were reprobed with α-tubulin antibodies to ensure equal sample loading. The expression of the apoptotic markers was increased with respect to α-tubulin levels in both cell lines treated with the fucoidan fractions. Band intensities were quantified using Quantity One (version 4.6.5., BioRad) and the histograms were plotted with GraphPad Prism.

altering the expression of mitochondria-associated proteins, cell cycle regulatory proteins, and transcription factors (Ye et al., 2005). Aisa et al. reported that fucoidan induced apoptosis in HS-Sultan cells by activating caspase-3 (Aisa et al., 2005). They were found to induce apoptosis in colon cancer cells by activating caspase-9, leading to the activation of caspase-3 (Hyun et al., 2009; Kim et al., 2010). Miyamoto et al. also reported fucoidan-induced, caspase-dependent apoptosis in human breast cancer cells (Yamasaki-Miyamoto et al., 2009). In the present study, we found that fucoidan induced activation of caspase-8, -9, and -3, as well as the concomitant cleavage of PARP-1. These results were further substantiated by the works of Boo et al. Yang et al., and Park et al. (Boo et al., 2011; Park et al., 2013; Yang et al., 2013). All of these data suggest that fucoidan-induced apoptosis is caspase-dependent and seems to involve both extrinsic and intrinsic pathways. The transcription factor NFκB is implicated in various stages of cancer progression, as it regulates genes associated with tumor invasion, metastasis, and chemoresistance. These genes include pro-metastatic, pro-angiogenic, and antiapoptotic genes (Sohma et al., 2011). NFκB is generally maintained in its inactive form in the cytoplasm by its inhibitor, IκB. When IκB is phosphorylated by the IKK family of kinases, NFκB dimers are released from its inhibitor, followed by the proteosomal degradation of IκB; the most predominantly occurring and transcriptionally active NFκB dimer is the p65/p50 heterodimer (Garcia-Pineres et al., 2001; Nakanishi and Toi, 2005). Nuclear translocation of NFκB subsequently activates the transcription of many genes associated with oncogenesis and tumor progression. Several studies have revealed that inhibition of NFκB activation sensitizes tumor cells to apoptosis (Arlt et al., 2001; Guo et al., 2004; Mabuchi et al., 2004; Wang et al., 1999). This emphasizes the fact that NFκB can be used as a potential target for cancer therapy. Although fucoidan is report to induce apoptosis via the activation of caspases, the genetic alterations underlying these mechanisms have thus far not been investigated in PC. It has been demonstrated that Rel A, the p65 subunit of NFκB, is constitutively activated in human PC cells, but not in normal pancreatic tissues (Wang et al., 1999). The resistance of PC cells to chemotherapy has been positively correlated with the constitutive activation of the transcription factor NFκB (Baldwin, 2001; Mayo and Baldwin, 2000; Orlowski and Baldwin,

in the bioactivities between the fractions. These observations corroborate with the findings of Ye et al. (2008), who also reported the effect of sulfation on the antitumor properties of algal polysaccharides. Interestingly, the antiproliferative potential of fucoidan fractions did not follow the same trend in Panc-1 cells. This finding indicates that the antitumor activity of fucoidans from the same source varies according to cell type. Brennan et al. (1995) proposed that ester sulfates mediate cellular recognition by binding to specific cell surface receptors. Hence, it may be suggested that the difference in the inhibitory potential between MiaPaCa-2 and Panc-1 cells could arise due to the variations in the sulfate content. Fukahori et al. Athukorala et al., Jiang et al. and Costa et al. have also reported selective inhibition of tumor cells by fucoidans (Athukorala et al., 2009; Costa et al., 2011; Fukahori et al., 2008; Jiang et al., 2010). Apoptosis is an important hallmark of chemotherapy-induced cancer cell death, characterized by nuclear fragmentation and chromatin condensation (Frankfurt and Krishan, 2003). Fucoidans have been previously shown to induce apoptosis in human colon carcinoma cells (Hyun et al., 2009; Kim et al., 2010), cervical cancer cells, mucoepidermoid carcinoma cells (Lee et al., 2014), human leukemia U937 cells (Park et al., 2013), and human hepatocarcinoma cells (Yang et al., 2013). However, the concentration of fucoidans used in the above studies was much higher than that used in the present study. Caspases play a critical role in mediating apoptotic cell death. They are classified as either initiator or effector caspases, based on their mode of activation. Upon activation, initiator caspases can cleave and activate effector caspases (Li and Yuan, 2008). Caspase-dependent apoptosis follows two independent pathways. The extrinsic pathway is initiated by transmembrane death receptor interactions and is characterized by the activation of caspase-8, which in turn activates the effector caspase-3 (Elmore, 2007). In the intrinsic pathway, mitochondrial depolarization triggers the activation of caspase-9, followed by caspase-3 (Szegezdi et al., 2003; Wei et al., 2000). Both pathways converge at the activation of caspase-3, which is responsible for the apoptotic morphology through the cleavage of various cellular substrates, such as PARP. Cleaved PARP is therefore generally used as an apoptotic marker, in addition to cleaved caspases (Kaufmann et al., 1993). Several studies have shown that fucoidans induce extrinsic and intrinsic pathways by

5

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Fig. 4. Fucoidan regulates the NFκB signalling transcriptome in pancreatic cancer cells. Histograms of QPCR profiling comparison analysis show regulation of NFκB signalling transcriptome (88 genes associated with NFκB signalling) by fucoidan fraction F5 in MiaPaCa-2, Panc-1, Panc-3.27, and BxPC-3 cells. The ΔΔct values were calculated by normalizing the gene expression levels to the expression of the housekeeping genes. The normalized data were then compared between groups, and the relative expression level of each gene was expressed as fold change. Fucoidan elicited cell-line-dependent and cell-lineindependent downregulation of NFκB signalling molecules in pancreatic cancer cells.

ingenuity pathway analysis (IPA) identified more than 252 different canonical signalling pathways in which these molecules have critical and functional roles. From this analysis, we have constructed an overlapping network of the top 50 canonical signalling pathways (Figure S2). Interestingly, such a construction of overlapping signalling networks revealed tight associations between pancreatic adenocarcinoma signalling and NFκB signalling, along with another 32 signalling pathways (Figure S2). In addition to their defined roles and association in canonical signalling, we also evaluated their intricate roles in molecular and cellular functions (Table S2). Altogether, these 24 fucoidantargeted molecules are critically involved in more than 500 annotated cellular functions, which clearly shows their all-inclusive role in the orchestration of disease progression. For ease of presentation, only a list of the top 25 cellular functions identified by the ingenuity pathway analysis is included in Table S2. All of the relationship analyses for the 24 fucoidan-targeted molecules were performed by strictly adhering to the confidence equivalent of ‘experimentally observed’. Their top roles,

2002). To that end, the present study investigated the molecular mechanisms of fucoidan in regulating NFκB-mediated signal transduction. The PCR-based array revealed that most NFκB-related genes were downregulated in PC cells. Interestingly, three genes, BCl10, IFNA1, and TNFRSF10B, were inhibited across all four cell lines investigated. In addition, we observed that another 21 genes, including BCL2L1, BCL3, BIRC2, BIRC5, CCL2, CHUK, F2R, FADD, HMOX1, IL12A, IRAK1, JUN, LTA, MAP3K1, NLRP12, NOD1, PPM1A, TLR4, TLR9, TMED4, and TRADD, showed cell-line-independent inhibition in at least three cell lines (see Fig. 4). Inhibition of these anti-apoptotic molecules with fucoidan treatment is consistent, at least in part, with findings from previous studies (Zhang et al., 2011). Although it is not feasible and is beyond the scope of this manuscript to discuss the functional relevance of each of these 24 fucoidaninhibited transcripts in disease progression, it is pertinent to mention that these molecules heavily contribute to the tightly inter-regulated overlapping networks of diseases and function (Figure S1). Further, 6

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Khanbolooki et al. (Fujioka et al., 2003; Khanbolooki et al., 2006). Lee et al. reported that fucoidan from Fucus vesiculous attenuated NFκB activation by inhibiting IκB degradation and p65 nuclear translocation in human lung cancer cells (Lee et al., 2012). Furthermore, consistent with the observations of Lee et al. and Shu et al. fucoidan inhibited the nuclear translocation of p65, thereby inhibiting the DNA binding activity of NFκB (Lee et al., 2012; Shu et al., 2015). However, transcriptional activation of NF-κB does not always follow p65 nuclear translocation (Campbell et al., 2001; Chen et al., 2001, 2002; Wang and Baldwin, 1998; Zhong et al., 1997). NFκB-dependent luciferase reporter assay was performed to confirm the inhibition of NFκB transcriptional activity with fucoidan treatment in PC. Recently, there has been a surge in the studies concerning NFκB as a therapeutic target in cancer treatment (Pramanik et al., 2018). The NFκB inhibiting activity of fucoidans holds promise in the development of effective drugs for pancreatic cancer. In contrast to the function of NFκB, activation of p53 induces apoptosis in PC (Izetti et al., 2014). While NFκB can promote cell survival and resistance to apoptosis, p53 initiates cell cycle arrest and apoptosis (Vogelstein et al., 2000). There exists a functional crosstalk between p53 and NFκB transcription factors that can result in alterations in the gene expression patterns in tumor cells (Bisio et al., 2013). NFκB negatively regulates p53 stability by increasing the amount of murine double mutant 2 (Mdm2). IKKβ-mediated NFκB activation increases the levels of Mdm2, resulting in the inhibition of p53-induced apoptosis (Rocha et al., 2005; Tergaonkar et al., 2002). Furthermore, activation of either p53 or NFκB depends on the relative nuclear levels of Rel/NFκB and p53 competing for the co-activator protein p300 and CREB binding protein (CBP) (Ravi et al., 1998; Wadgaonkar et al., 1999; Webster and Perkins, 1999). The nuclear level of p300/CBP is very low, and it plays a crucial role in determining the transcriptional activation of p53 and NFκB. Nevertheless, compared with NFκB, p53 forms a stable complex with p300/CBP, favoring apoptosis and inhibiting NFκB activation (Avantaggiati et al., 1997; Lill et al., 1997). Reducing the expression of p53 increases NFκB activation, and vice versa. The fact that p53 is mutated in more than 50% of PC tissues and the constitutive activation of NFκB support the inverse correlation between these two transcription factors (Li et al., 2004; Ruggeri et al., 1997). Our results demonstrated a significant induction of p53 in PC cells in response to fucoidan treatment and revealed that fucoidan intervenes in the NFκB-p53 cross talk by targeting the DNA binding and transcriptional activity of NFκB, leading to NFκB inhibition-dependent p53 activation, thereby orchestrating apoptosis in PC cells. However, the bioavailability and the pharmacokinetics of the fucoidans have not been investigated extensively. Reports suggest that very small quantities of fucoidan can be absorbed upon oral ingestion (Fitton et al., 2015b). The decreased bioavailability could be due to their higher carbohydrate content and nanoencapsulation could increase its bioavailability as reported by several investigators (Kimura et al., 2013; Shofia, 2018). Hence, studies on the bioavailability and efficacy of fucoidans in animal models are highly warranted.

Fig. 5. Fucoidan inhibits NFκB activation in pancreatic cancer cells. Fucoidan fraction F5 suppressed the DNA binding and transcriptional activation of NFκB in MiaPaCa-2 cells. Electrophoretic mobility shift assay showed that F5 inhibited the DNA binding activity of both constitutively expressed and TNF-α induced NFκB (A). Exogenous TNF-α enhanced the DNA binding activity of NFκB, expressed as band intensity, and was significantly reduced by fraction F5 in a dose-dependent manner (B). Fraction F5 also produced significant reduction in the NFκB-dependent luciferase activity in MiaPaCa-2 cells (C). Furthermore, this fraction stabilized IκBα in the cytoplasm, while maintaining the cytosolic levels of p65 (D).

Fig. 6. Fucoidan mediates NFκB-associated p53 regulation. Fucoidan fractions significantly upregulated p53 expression in MiaPaCa-2 cells (A). Fraction F5 restored the expression levels of p53 in cells ectopically expressing p65 (B), thereby favoring apoptosis.

4. Conclusion

percent overlap, number of molecules involved, and their significance in each of the categories are summarized in Table S3. In addition, fucoidan effectively downregulated the expression of members of NFκB family, the activation of which promotes tumor progression. This finding is consistent with EMSA results, which revealed a marked inhibition in TNFα-mediated NFκB activation in PC cells. As discussed earlier, NFκB is rendered inactive by its association with IκB (Li et al., 2004; Oeckinghaus and Ghosh, 2009; Veeraraghavan et al., 2011b). Immunoblotting revealed clearly increased expression of IκBα, indicating that TNFα-induced NFκB activation was inhibited by the downregulation of IKK, which did not phosphorylate IκBα in PC cells. Inhibition of IκBα degradation associated with the suppressed NFκB activation was also observed in PC cells by Fujioka et al. and

In conclusion, fucoidan from Turbinaria conoides inhibits the growth of PC cells and induces apoptosis through activation of caspase-dependent intrinsic and extrinsic pathways. In addition, fucoidan negatively regulates NFκB signalling by inhibiting constitutive and TNF-αmediated NFκB activation. Fucoidan also increases the expression of tumor suppressor p53 in an NFκB-inhibition-dependent manner, which induces apoptosis in PC cells. Taken together, these findings indicate that fucoidan fraction F5 from Turbinaria conoides is a potential candidate for the development of anti-pancreatic tumor therapy. The authors acknowledge the limitations of the current study, including the need for pharmacokinetics and preclinical studies with appropriate spontaneous pancreatic cancer mouse models to identify the efficacy of 7

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the fucoidan fractions in this setting. However, such preclinical exhaustive evaluation studies are beyond the scope of the current manuscript, as the outcomes of the manuscript are focused on mechanistic insights into how the fucoidan fraction exerts PC cell killing. However, such preclinical anti-PC studies, along with pharmacokinetics studies, are currently planned and/or are underway in our laboratory.

determine the dose-dependent effects. For the inhibitory kinetics of individual fractions, the effect was examined after 24 and 72 h. All samples were assayed in triplicate. Two-way ANOVA with Bonferroni's post-hoc test was performed for group-wise comparisons (GraphPad PRISM 6.0). A P value of p < 0.05 is considered statistically significant.

5. Experimental

5.5. Immunoblotting

5.1. General experimental procedures

MiaPaCa-2 and Panc-1 cells were treated with 5 μg/ml of fucoidan fractions F2, F3, F4, and F5 for 24 h. Total cellular protein isolation, quantitation, and immunoblotting were performed as described in our earlier studies (Natarajan et al., 2002). The membranes were washed and re-labelled with corresponding HRP-conjugated secondary antibody for 45 min. The membranes were stripped with stripping buffer containing 100 mM β-mercaptoethanol and 2% SDS at 50 °C for 30 min, and were reprobed with mouse anti-α-tubulin antibody (Sigma, MO, USA). Band intensities were quantified using Quantity One (version 4.6.5., BioRad) and the group-wise comparisons were plotted with GraphPad Prism.

Genetically diverse human pancreatic adenocarcinoma cell-lines Panc-1, MiaPaCa-2, Panc-3.27 and BxPC-3 were procured from ATCC (Manassas, VA, USA). Cells were cultured and maintained as described earlier (Aravindan et al., 2017; Delma et al., 2015). The p53 mutation status of the cell lines (MiaPaCa-2: homozygous, c.742C > T; Panc-1: homozygous, c.818G > A; BXPC-3: homozygous, c.659A > G; Panc 3.27: homozygous, c.376-1G > T) used was obtained from www.atcc. org (Web reference). For passaging and for all experiments, the cells were detached using 0.25% trypsin/1% EDTA, resuspended in complete medium, counted electronically using a Countess automated cell counter (Carlsbad, CA, USA), and incubated in a 95% air/5% CO2 humidified incubator. Rabbit anti-caspase 3, rabbit anti-caspase 8, rabbit anti-caspase 9, anti-p65, anti-p53 and goat anti-rabbit IgG-HRP antibodies were obtained from Santa Cruz Biotechnology, CA, USA. Antibodies against IκBα and PARP were obtained from Cell Signalling Technology, MA, USA.

5.6. NFκB signalling pathway transcriptome QPCR profiling

The brown alga, Turbinaria conoides (J. Agardh) Kützing was collected from Tuticorin coast, South-east coast of India during September–October 2008. Fucoidan was extracted from the brown alga Turbinaria conoides (J. Agardh) Kützing by hot water extraction for 16 h at 95 °C as described by Noseda and colleagues (Duarte et al., 2001), with slight modifications. The filtrate obtained was precipitated with absolute ethanol and dialyzed against distilled water, then lyophilized (Lark Innovative, TN, India). The brown powder that was obtained is the crude fucoidan extract (Delma et al., 2015). Our recent investigations clearly demonstrated the anti-PC potential of the T. conoides fucoidan crude extract (Delma et al., 2015). Crude fucoidan was then fractionated by anion exchange chromatography. It was applied on a DEAE cellulose column (2 × 18 cm) linked to a FPLC system (Biorad, USA) equilibrated with 50 mM sodium acetate buffer (pH 5.0). The column was eluted with a linear gradient of 0–3.0M NaCl in the same buffer. The flow rate was maintained at 0.5 ml/min. Fractions of 5 ml were collected and assayed for total sugars (Masuko et al., 2005). Altogether, five fractions (F1, F2, F3, F4, and F5) containing polysaccharides were pooled, dialyzed, and lyophilized.

MiaPaCa-2, Panc-1, Panc-3.27 and BxPC-3 cells were treated with 5 μg/ml of fucoidan fraction F5 for 3 h. Cells were harvested and lysed with Tripure reagent (Roche). Total RNA extraction and real-time QPCR profiling were performed as described earlier (Aravindan et al., 2013b) using a custom-made human NFκB signalling pathway profiler (Realtimeprimers.com, Elkins Park, PA). We started with this highly selective QPCR profiler instead of an all-encompassing gene array because the selected genes entail a well-characterized profile governing the NFκB pathway. This selection facilitates interpretation of data, simplifying data acquisition and analysis, and avoids genes that are not functionally characterized. QPCR profiling was done in the 96-well PCR array plates equipped with a panel of primer sets for 88 genes associated with NFκB signalling (Table S1). Each profiling plate was equipped with eight house-keeping genes: β-actin, β-2-microglobulin, glyceraldehyde phosphate dehydrogenase (GAPD), β-glucuronidase, hypoxanthine phosphoribosyl transferase 1(HPRT1), phosphoglycerate kinase 1 (PGK1), ribosomal protein L13a, and peptidyl propyl isomerase A. The ΔΔct values were calculated by normalizing the gene expression levels to the expression of the housekeeping genes. The normalized data were then compared between groups, and the relative expression level of each gene was expressed as fold change. When comparing each gene's signal intensity between groups, we used a ≥2 fold increase or decrease to represent “stringent” criteria for upregulation or downregulation, and an increase/decrease of < 2 fold to represent “less stringent” criteria. Classifying gene regulation criteria in this way can provide an index of reliability of the gene expression data (Veeraraghavan et al., 2011b).

5.3. Chemical characterization of fucoidan fractions

5.7. Electrophoretic mobility shift assay (EMSA)

The total polysaccharide content of the five fractions was estimated using the microplate method of Masuko and colleagues (Masuko et al., 2005). The uronic acid content was determined by following the protocol described in earlier studies. Sulfate content was estimated by the benzidine method using sodium sulfate as the standard (Terho and Hartiala, 1971).

MiaPaCa-2 cells were treated with 1, 2, 5, and 10 μg/ml of fucoidan fraction F5 for 12 h. TNF-α at a concentration of 1 ng/ml was added and incubated for 1 h. Nuclear protein extraction and electrophoretic mobility shift assay (EMSA) assessing the NFκB DNA binding activity was performed as described in our earlier studies (Natarajan et al., 2002; Veeraraghavan et al., 2011a).

5.4. MTT assay

5.8. Luciferase reporter assay

The effect of fucoidan fractions (1 through 5) on the survival of PC cell lines was evaluated by MTT assay, as described in our earlier studies (Aravindan et al., 2013a; Delma et al., 2015). A wide range of concentrations (50, 25, 12.5, 6.25 and 3.125 μg/ml) were used to

The effect of the fucoidan fraction F5 on NFκB transcriptional activity was determined by Luciferase reporter assay, as described previously (Aravindan et al., 2013a). The pNFκB-Luc-transfected MiaPaCa2 cells were treated with 5 μg/ml of fraction F5 for 24 h. The

5.2. Extraction and fractionation of fucoidan by anion exchange chromatography

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luminescence emitted was read using Synergy 4 Multidetection Microplate Reader (Biotek Instruments Inc., Vermont, USA). The data were subjected to ANOVA with Tukey's Post-hoc correction. A P value of < 0.05 is considered statistically significant.

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Funding The research was partly supported by funding from the Department of Biotechnology, Ministry of Science and Technology, Government of India (BT/PR11535/AAQ/03/431 to ST). Declarations of interests None. Acknowledgements The authors thank Dr. T.T. Ajith Kumar, Principal Scientist, PMFGR Centre, ICAR-National Bureau of Fish Genetic Resources, for providing the algal biomass and Dr. Ganesan, Scientist, CSIR-CSMCRI for seaweed identification. The authors also thank Dr. M. Arumugam, Assistant Professor, CAS in Marine Biology, Annamalai University for helping with the chemical analyses and Prof. T. Balasubramanian, Former Dean, Faculty of Marine Sciences, Annamalai University for providing institutional facilities. The authors acknowledge the OUHSC Editorial Review of Scientific Communications for help in reviewing the manuscript. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.phytochem.2019.112078. References Aisa, Y., Miyakawa, Y., Nakazato, T., Shibata, H., Saito, K., Ikeda, Y., Kizaki, M., 2005. Fucoidan induces apoptosis of human HS-sultan cells accompanied by activation of caspase-3 and down-regulation of ERK pathways. Am. J. Hematol. 78, 7–14. Ale, M.T., Maruyama, H., Tamauchi, H., Mikkelsen, J.D., Meyer, A.S., 2011. Fucosecontaining sulfated polysaccharides from brown seaweeds inhibit proliferation of melanoma cells and induce apoptosis by activation of caspase-3 in vitro. Mar. Drugs 9, 2605–2621. Aravindan, S., Delma, C.R., Thirugnanasambandan, S.S., Herman, T.S., Aravindan, N., 2013a. Anti-pancreatic cancer deliverables from sea: first-hand evidence on the efficacy, molecular targets and mode of action for multifarious polyphenols from five different brown-algae. PLoS One 8, e61977. Aravindan, S., Natarajan, M., Herman, T.S., Aravindan, N., 2013b. Radiation-induced TNFalpha cross signaling-dependent nuclear import of NFkappaB favors metastasis in neuroblastoma. Clin. Exp. Metastasis 30, 807–817. Aravindan, S., Ramraj, S., Kandasamy, K., Thirugnanasambandan, S.S., Somasundaram, D.B., Herman, T.S., Aravindan, N., 2017. Hormophysa triquerta polyphenol, an elixir that deters CXCR4- and COX2-dependent dissemination destiny of treatment-resistant pancreatic cancer cells. Oncotarget 8, 5717–5734. Aravindan, S., Ramraj, S.K., Somasundaram, S.T., Aravindan, N., 2015a. Novel adjuvants from seaweed impede autophagy signaling in therapy-resistant residual pancreatic cancer. J. Biomed. Sci. 22, 28. Aravindan, S., Ramraj, S.K., Somasundaram, S.T., Herman, T.S., Aravindan, N., 2015b. Polyphenols from marine brown algae target radiotherapy-coordinated EMT and stemness-maintenance in residual pancreatic cancer. Stem Cell Res. Ther. 6, 182. Arlt, A., Vorndamm, J., Breitenbroich, M., Folsch, U.R., Kalthoff, H., Schmidt, W.E., Schafer, H., 2001. Inhibition of NF-kappaB sensitizes human pancreatic carcinoma cells to apoptosis induced by etoposide (VP16) or doxorubicin. Oncogene 20, 859–868. Atashrazm, F., Lowenthal, R.M., Woods, G.M., Holloway, A.F., Dickinson, J.L., 2015. Fucoidan and cancer: a multifunctional molecule with anti-tumor potential. Mar. Drugs 13, 2327–2346. Athukorala, Y., Ahn, G., Jee, Y., Kim, G., Kim, S., Ha, J., Kang, J., Lee, K., Jeon, Y., 2009. Antiproliferative activity of sulfated polysaccharide isolated from an enzymatic digest of Ecklonia cava on the U-937 cell line. J. Appl. Phycol. 21, 307–314. Avantaggiati, M.L., Ogryzko, V., Gardner, K., Giordano, A., Levine, A.S., Kelly, K., 1997. Recruitment of p300/CBP in p53-dependent signal pathways. Cell 89, 1175–1184. Baldwin, A.S., 2001. Control of oncogenesis and cancer therapy resistance by the transcription factor NF-kappaB. J. Clin. Investig. 107, 241–246. Bisio, A., Zámborszky, J., Zaccara, S., Lion, M., Tebaldi, T., Ciribilli, Y., Inga, A., 2013. Abstract 746: functional crosstalk between the p53 and NF-kB transcription factors.

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