Research ArticleExperimental Studies

Protective Role of Fucoidan on Cisplatin-mediated ER Stress in Renal Proximal Tubule Epithelial Cells

HYUNG JOO KIM, YEO MIN YOON, JUN HEE LEE and SANG HUN LEE
Anticancer Research October 2019, 39 (10) 5515-5524; DOI: https://doi.org/10.21873/anticanres.13744

Abstract

Background/Aim: Administration of cisplatin in cancer patients is limited by the kidney-related adverse effects; however, a protective strategy is absent. We hypothesized that fucoidan protects the proximal tubule epithelial (TH-1) cells against the effects of cisplatin. Materials and Methods: To assess the effect of fucoidan, its effect on reactive oxygen species (ROS) formation, endoplasmic reticulum (ER) stress response, DNA damage response (DDR), apoptosis, and cell-cycle arrest in TH-1 cells was investigated. Results: Cisplatin increased the accumulation of ROS, leading to excessive ER stress. In presence of cisplatin, treatment of TH-1 cells with fucoidan significantly reduced the ER stress by maintaining the complex of GRP78 with PERK and IRE1α. In particular, fucoidan enhanced the antioxidative capacity through up-regulation of PrPC. Furthermore, fucoidan suppressed cisplatin-induced apoptosis and cell-cycle arrest, whereas silencing of PRNP blocked these effects of fucoidan. Conclusion: Fucoidan may be a potential adjuvant therapy for cancer patients treated with cisplatin as it preserves renal functionality.

Cisplatin or cis-diamminedichloroplatinum (CDDP, cis-[PtCl2(NH3)2]) is one of the most widely used chemical agents in anti-cancer chemotherapy (1). However, since the proximal tubule is extremely susceptible to accumulation of cisplatin, acute kidney injury (AKI), the abrupt failure of kidney function, is a frequent side effect of cisplatin therapy, which occurs in 25%-35% of patients receiving the treatment (2, 3). Cisplatin is expected to damage DNA and initiate the DNA damage response (DDR) pathway. The heightened activation of the response causes cell cycle arrest and apoptosis by regulating downstream effectors such as P53, ATM, and ATR (4, 5). Cisplatin is also known to induce ER stress and initiate unfolded protein response (UPR) (6). When ER stress is not mitigated and homeostasis is not restored, the downstream effectors of the UPR stimulate the formation of excess of ROS and disrupt calcium homeostasis, inducing caspase-3 mediated apoptosis and P53-dependent cell cycle arrest (7, 8). In fact, many studies have revealed that excess generation of ROS by renal-infiltrating or resident cells are determinant factors for inducing the anticancer drug side effects (9, 10).

Fucoidan, sulfated polysaccharides extracted from brown algae, display antibacterial, antiviral, antioxidant, and antitumor activities (11, 12). Multiple studies have revealed that fucoidan plays an important pro-survival role against ROS generation via up-regulation of antioxidative enzymes (13, 14). As an antioxidant, fucoidan limits oxidative stress by scavenging superoxide radicals, facilitating the expression of superoxide dismutase (SOD), and suppressing the transforming growth factor β (TGF-β)/SMAD pathway, which prevents ROS generation in cancer cells and ROS release into tumor microenvironment (15-17). However, the underlying mechanism by which fucoidan protects against ER stress remains elusive and needs further investigations. To clarify the effect of fucoidan on kidney cells under ER stress conditions, we investigated whether fucoidan protects against cisplatin-induced ER stress in human renal proximal tubule epithelial cells through up-regulation of PrPC.

Materials and Methods

Human renal proximal tubule epithelial culture. Human renal proximal tubule epithelial (TH-1) cells were obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were maintained in Minimum Essential Medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% (v/v) fetal bovine serum (Thermo Fisher Scientific) and 100 U/ml penicillin/streptomycin (Thermo Fisher Scientific). Cells were incubated in a humidified incubator at 37°C and 5% CO2.

Western blot assay. Cell lysates (20 μg protein) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins were transferred to polyvinylidene fluoride membranes (Sigma Aldrich, St. Louis, MO, USA). The membranes were blocked with 5% skimmed milk for 1 h and incubated with the appropriate primary antibodies against protein kinase R-like endoplasmic reticulum kinase (PERK), phospho-PERK (p-PERK), eukaryotic initiation factor 2-alpha (eIF2α), p-eIF2α, activating transcription factor 4 (ATF4), inositol-requiring protein 1α (IRE1α), p-IRE1α, c-Jun N-terminal kinase (JNK), p-JNK, CCAAT-enhancer-binding protein homologous protein (CHOP), B-cell lymphoma 2 (BCL-2), BCL-2-associated X protein (BAX), cleaved caspase-3, cleaved poly(ADP ribose) polymerase-1 (PARP-1), p53, p-p53, ataxia telangiectasia mutated (ATM), p-ATM, cellular prion protein (PrPC), manganese-dependent superoxide dismutase (MnSOD), and β-actin. All antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The membranes were then washed, and the primary antibodies were detected using goat anti-rabbit IgG or goat anti-mouse IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology). The bands were visualized using enhanced chemiluminescence (Sigma Aldrich).

Dihydroethidium (DHE) staining. DHE (Sigma) was used to measure superoxide anions in TH-1 cells. TH-1 cells were trypsinized to obtain cell suspensions and exposed to DHE (10 μM) for 30 min at 37°C. After washing with PBS three times, samples were measured by fluorescence-activated cell sorting (FACS, Sysmex, Kobe, Japan) analysis. DHE-positive cells were identified using Flowing Software (DeNovo Software, Los Angeles, CA, USA).

Immunoprecipitation. TH-1 cells were lysed with a lysis buffer (1% Triton X-100 in 50 mM Tris-HCl [pH 7.4] consisting of 150 mM NaCl, 5 mM EDTA, 2 mM Na3VO4, 2.5 mM Na4PO7, 100 mM NaF, and protease inhibitors). Each protein sample (300 μg) was mixed with anti-GRP78 antibody (Santa Cruz Biotechnology) at 4°C overnight. The samples were mixed with Protein A/G PLUS-Agarose Immunoprecipitation Reagent (Santa Cruz Biotechnology) and incubated for 12 h at 4°C, after which the beads were washed four times with PBST (Phosphate Buffered Saline with Tween 20). The bound proteins were released from the beads by boiling in SDS-PAGE sample buffer for 5 min. The precipitated proteins were visualized by western blotting with anti-PERK antibody or anti-IRE1α antibody (Santa Cruz Biotechnology).

Catalase activity. TH-1 cellular proteins were extracted using RIPA extraction buffer (Thermo Fisher Scientific). Catalase activity was measured using a Catalase Assay Kit (Sigma Aldrich) according to the manufacturer's instructions. Protein (50 μg) samples were reacted with 200 mM H2O2 for 3 min, and then stop buffer was added. Then, the color reagent containing 150 mM potassium phosphate buffer, pH 7.0, containing 0.25 mM 4-aminoantipyrine and 2 mM 3,5-dichloro-2-hydroxybenzenesulfonic acid was added for 15 min and the optical density was measured by microplate reader (BMG labtech, Ortenberg, Germany) at 520 nm. The calculation of catalase activity was determined according to the manufacturer's instruction.

Superoxide dismutase activity. TH-1 cellular proteins were extracted using RIPA extraction buffer (Thermo Fisher Scientific). SOD activity was measured using a SOD activity kit (Enzo, Basel, Switzerland). Protein samples were added onto each well of 96-well plate in aliquots of 25 μl containing 40 μg. Then 150 μl of the master mix, containing WST-1 reagent and Xanthine oxidase, was added onto each well. After xanthine solution was added (25 μl/well), the optical density was measured every minute for 15 min using microplate reader (BMG labtech) at 450 nm. The calculation of SOD activity was determined following the manufacturer's instruction.

Propidium iodide (PI)/Annexin V flow cytometric analysis. Apoptosis of TH-1 cells was assessed using Cy-Flow Cube 8 Flow Cytometer (Partec, Münster, Germany) after staining the cells with PI and Annexin V-FITC for 30 min (De Novo Software, Los Angeles, CA, USA). PI or Annexin V positivity was analyzed using standard FCS Express Software (De Novo Software).

Cell proliferation assay. TH-1 cells were cultured in 96-well culture plates (3000 cells/well) and treated with 5-bromo-2’-deoxyuridine (BrdU), which is incorporated into the newly synthesized DNA of proliferating cells, and then these cells were assessed by a BrdU ELISA colorimetric kit (Roche, Basel, Switzerland). The cells were incubated with, 100 μg/ml BrdU at 37°C for 3 h. The cell media were replaced with BrdU labeling solution and FixDenat solution (200 μl/well) for 30 min. Next, an anti-BrdU antibody conjugated with peroxidase (100 μl) was added in each well at room temperature for 90 min. To detect the immune complexes between the antibody and the DNA 100 μl of a tetramethyl-benzidine (TMB) substrate solution was added at room temperature for 20 min. Absorbance of the samples was measured using a microplate reader (BMG Labtech) at 370 nm.

Statistical analysis. Results were expressed as mean±standard error of the mean (SEM). The significance between groups was assessed by two-tailed student's t-test or by one- or two-way analysis of variance (ANOVA). Comparison between three or more groups was conducted using Dunnett's or Tukey's post-hoc test. Data were considered significantly different at p-value <0.05.

Results

Cisplatin induced ER stress through ROS generation. To explore the effect of cisplatin on the production of ROS in TH1 cells, flow cytometry analysis following DHE staining was performed in cisplatin-treated TH1 cells. Treatment with cisplatin significantly increased the level of ROS (Figure 1A). In addition, cisplatin significantly increased the activation of DDR-associated proteins, such as p-53 and p-ATM, in a time dependent manner (Figure 1B and C). To further investigate whether cisplatin-induced ROS accumulation induces ER stress, the activation of UPR signaling pathway proteins, including IRE1α, JNK, p38, and CHOP, was assessed by western blotting in TH1 cells. After treatment of TH1 cells with cisplatin for different time points (0, 6, 12, and 24 h), the expression of p-IRE1α, p-JNK, p38, and CHOP was significantly increased in a time dependent manner (Figure 1D-G). These results indicated that cisplatin induces ER stress and increased accumulation of ROS in renal proximal tubule epithelial cells that display DDR and UPR responses.

Figure 1.
Figure 1.

Cisplatin induced ER stress through increased formation of ROS, which activated DNA damage response (DDR) and Unfolded protein response (UPR). (A) DHE/ROS positive TH-1 cells treated with or without cisplatin were measured by FACS. (B, C) Activation of DDR associated proteins, p-P53 and p-ATM, was measured in TH-1 cells treated with cisplatin for 0, 24, 48, and 72 h. The expression levels were determined by densitometry relative to p53 or ATM, respectively. Values represent the mean±SEM. *p<0.05 and **p<0.01 vs. untreated TH-1 cells. (D-G) Expression and activation of ER stress markers, GRP78, p-PERK, p-eIF2α, ATF4, p-IRE1α, p-JNK, p-P38, and CHOP, were detected in TH-1 cells treated with cisplatin for 0, 24, 48, and 72 h. The expression levels were determined by densitometry relative to β-actin, PERK, eIF2α, IRE1α, JNK, or P38, respectively. Values represent the mean±SEM. *p<0.05 and **p<0.01 vs. untreated TH-1 cells.

Fucoidan inhibited the activations of the UPR and DNA repair pathways by mitigating ER stress. To investigate the protective effect of fucoidan against cisplatin-induced ER stress, we analyzed the binding of GRP78, which is a chaperon protein, with PERK, after treatment of TH1 cells with cisplatin in the presence or absence of fucoidan. Cisplatin significantly decreased the formation of GRP78/PERK complexes, whereas fucoidan suppressed the dissociation of the GRP78/PERK complex (Figure 2A). In addition, cisplatin significantly induced the expression of ER stress-associated proteins, including GRP78, p-PERK, p-eIF2α, and ATF4, in TH1 cells, but treatment with fucoidan reversed the effect of cisplatin (Figure 2B and C). Cisplatin also significantly blocked the formation of GRP78/IRE1α complex (Figure 2D). However, fucoidan significantly enhanced the formation of the GRP78/IRE1α complex in presence of cisplatin, leading to inhibition of cisplatin-induced ER stress by decreasing the expression of UPR response associated proteins, IRE1α, JNK, P38, and CHOP (Figure 2E and F).

Figure 2.
Figure 2.

Fucoidan suppressed the activation of the UPR by mitigating ER stress. (A) Immunoprecipitation using anti-GRP78 and anti-PERK antibodies was analyzed after TH-1 cells, which were pretreated with or without fucoidan, were treated with cisplatin. The level of PERK binding to GRP78 was normalized to that of GRP78 alone. Values represent the mean±SEM. *p<0.01 vs. untreated TH-1 cells, ##p<0.01 vs. cisplatin treated TH-1 cells. (B) Expression and activation of ER stress markers, GRP78, p-PERK, p-eIF2α, and ATF4, were detected using western blot analysis after TH-1 cells, which were pre-treated with or without fucoidan, were treated with cisplatin. (C) The expression levels were determined by densitometry relative to β-actin, PERK, or eIF2α, respectively. Values represent the mean±SEM. *p<0.05 and **p<0.01 vs. untreated TH-1 cells, ##p<0.01 vs. cisplatin treatment of TH-1 cells. (D) Immunoprecipitation using anti-GRP78 and anti-IRE1α antibodies was analyzed after TH-1 cells, which were pretreated with or without fucoidan, were treated with cisplatin. The level of IRE1α binding to GRP78 was normalized to that of GRP78 alone. Values represent the mean±SEM. **p<0.01 vs. untreated TH-1 cells, ##p<0.01 vs. cisplatin treated TH-1 cells. (E) Expression and activation of other ER stress markers, IRE1α, p-JNK, p-P38, and CHOP, were detected using western blot analysis after TH-1 cells, which were pre-treated with or without fucoidan, were treated with cisplatin. (F) The expression levels were determined by densitometry relative to β-actin, IRE1α, JNK, or p38, respectively. Values represent the mean±SEM. *p<0.05 and **p<0.01 vs. untreated TH-1 cells, ##p<0.01 vs. cisplatin treatment of TH-1 cells.

Our results also showed that fucoidan was effective in limiting the DNA damage that arises from ROS-mediated oxidative stress. It is known that 4-PBA strongly inhibits ER stress and accumulation of ROS (18). Cisplatin treatment resulted in increased levels of DNA repair associated proteins, p-p53 and pATM, an effect that was reversed by 4-PBA (Figure 3A and B). In the same manner, fucoidan pretreatment of TH-1 cells resulted in decreased activation of the DNA repair pathway, indicating its effectiveness in mitigating the ER stress and suppressing ROS accumulation (Figure 3C and D).

Fucoidan exerts antioxidative effect by up-regulating PrPC expression. Previous studies have shown that PrPC reduced cellular ROS by up-regulating the major anti-oxidative enzyme known as mitochondrial antioxidant manganese superoxide dismutase (MnSOD) (19). To elucidate whether fucoidan increases the antioxidative effect in TH-1 cells via the expression of PrPC and MnSOD, their expression was assayed by western blotting. Fucoidan treatment significantly up-regulated the expression of PrPC and MnSOD in a time-dependent manner (Figure 4A and 4B). In addition, silencing of PrPC inhibited the fucoidan-induced increase in MnSOD levels (Figure 4C and D). In presence of cisplatin, the activities of catalase and SOD were significantly decreased in TH-1 cells (Figure 4E and F). However, fucoidan treatment in the presence of cisplatin resulted in the recovery of the activities of catalase and SOD, whereas knockdown of PRNP inhibited the activities of the anti-oxidant enzymes (Figure 4E and 4F). Furthermore, flow cytometry analysis for DHE staining indicated that the up-regulation of PrPC was responsible for cisplatin-induced ER stress-mediated ROS production (Figure 4G). These results showed that fucoidan augmented antioxidant activities in TH-1 cells, following cisplatin-induced ER stress, by increasing the expression of PrPC.

Figure 3.
Figure 3.

Fucoidan suppressed the DDR pathway by mitigating oxidative stress. (A) Activation of DDR markers, P53 and ATM, were analyzed by western blotting after TH-1 cells pretreated with or without 4-BPA were exposed to cisplatin. (B) The expression levels were determined by densitometry relative to P53 or ATM, respectively. Values represent the mean±SEM. **p<0.01 vs. untreated TH-1 cells, ##p<0.01 vs. cisplatin treatment of TH-1 cells. (C) Activation of DDR marker was analyzed by western blot after TH-1 cells pretreated with or without fucoidan were exposed to cisplatin (D) The expression levels were determined by densitometry relative to P53, or ATM, respectively. Values represent the mean±SEM. *p<0.05, and **p<0.01 vs. untreated TH-1 cells, ##p<0.01 vs. cisplatin treatment of TH-1 cells.

Fucoidan protected against cisplatin-induced apoptosis and cell cycle arrest through expression of PrPC. Our results indicated that the levels of the anti-apoptotic protein BCL2 were significantly decreased and the levels of pro-apoptotic proteins, BAX, cleaved caspase-3 (C-caspase-3), and cleaved PARP-1 (C-PARP-1), were increased by cisplatin treatment (Figure 5A and B). In the presence of cisplatin, treatment of TH-1 cells with fucoidan restored anti-apoptotic signaling, as indicated by the increased expression of BCL2 along with the decreased expression of the pro-apoptotic proteins, BAX, C-caspase-3, and C-PARP-1, whereas the silencing of PrPC prevented the effects of fucoidan (Figure 5A and B). Consistent with these results, flow cytometry analysis for PI/Annexin V staining showed that fucoidan protected from cisplatin-induced apoptosis through the expression of PrPC (Figure 5C and D). Moreover, our results showed that cisplatin induces cell cycle arrest by the down-regulation of cell cycle associated proteins, CDK2, cyclin E, CDK4, and cyclin D1 (Figure 6A-C). On the other hand, fucoidan treatment restored cell cycle progression to homeostatic level, by reversing the effect of cisplatin on the expressions of the cell cycle associated proteins (Figure 6A-C). As with the apoptosis, silencing of PrPC eliminated the protective effect of the fucoidan (Figure 6A-C). These findings indicated that the anti-apoptotic and pro-proliferation effects of fucoidan against cisplatin-mediated ER stress rely on the up-regulation of PrPC expression, which enhances the antioxidative capacity in the TH-1 cells.

Figure 4.
Figure 4.

Fucoidan-pretreated TH-1 cells showed enhanced antioxidant ability under cisplatin-induced ER stress by up-regulating PrPC expression. (A) Expression levels of PrPC and MnSOD in TH-1cells treated with fucoidan were measured at various time points (0, 6, 12, 24 h). (B) The expression levels were determined by densitometry relative to β-actin. Values represent the mean±SEM. *p<0.05 and **p<0.01 vs. untreated TH-1 cells. (C) Expression of PrPC and MnSOD were detected by western blot analysis after the treatment with fucoidan and si-PRNP silencing. (D) The expression levels were determined by densitometry relative to β-actin. Values represent the mean±SEM. **p<0.01 vs. untreated TH-1 cells, ##p<0.01 vs. cisplatin treatment of TH-1 cells, $$p<0.01 vs. fucoidan-treated TH-1 cells pretreated with si-PRNP. (E, F) Complex I and complex IV activities were analyzed by ELISA after treatment of fucoidan-pretreated TH-1 cells with si-PRNP. Values represent the mean±SEM. *p<0.05 vs. untreated TH-1 cells, #p<0.05 vs. cisplatin treatment of TH-1 cells, $p<0.05 vs. fucoidan-treated TH-1 cells pretreated with si-PRNP. (G) DHE staining was measured using FACS after treatment of fucoidan-pretreated TH-1 cells with si-PRNP. Values represent the mean±SEM. **p<0.01 vs. untreated TH-1 cells, ##p<0.01 vs. cisplatin treatment of TH-1 cells, $$p<0.01 vs. fucoidan-treated TH-1 cells pretreated with si-PRNP.

Figure 5.
Figure 5.

Fucoidan-mediated expression of PrPC decreased cisplatin-induced apoptosis. (A) Expression of BCL2, BAX, cleaved caspase 3, and cleaved PARP-1 was detected by western blot analysis after the treatment of fucoidan-pretreated TH-1 cells with si-PRNP. (B) The expression levels were determined by densitometry relative to β-actin. Values represent the mean±SEM. **p<0.01 vs. untreated TH-1 cells, ##p<0.01 vs. cisplatin treatment of TH-1 cells, $p<0.05, and $$p<0.01 vs. fucoidan-treated TH-1 cells pretreated with si-PRNP. (C) Apoptosis was analyzed using FACS after treatment of fucoidan-pretreated TH-1 cells with si-PRNP. (D) Values represent the mean±SEM. **p<0.01 vs. untreated TH-1 cells, ##p<0.01 vs. cisplatin treatment of TH-1 cells, $$p<0.01 vs. fucoidan-treated TH-1 cells pretreated with si-PRNP.

Discussion

The proximal tubule cells are susceptible to cisplatin as they contain the transporters and enzymes that readily uptake and metabolize cisplatin into nephrotoxin. Metabolically, cisplatin is metabolized to a cysteinyl-glycine-conjugate, to a cysteine-conjugate, and finally to a nephrotoxin thiol, by gamma-glutamyltransferase (20). The nephrotoxin inhibits the metabolism of halogenated alkenes, which induce mitochondrial dysfunction, the primary manifestation of cisplatin-induced cytotoxicity (21). As mitochondria are the source of reactive oxygen species, their malfunction has been shown to promote generation of reactive oxygen species (22). The oxidative environment within the ER lumen ensures proper protein folding. Abnormalities that lead to unfolding or misfolding of proteins produce ER stress and elicit UPR (23). Multiple studies have demonstrated ROS as the upstream signal of ER stress (24, 25). Moreover, ROS has been well recognized as agents that inflict damage on DNA and initiate DDR (26). Taken together, suppression of excess ROS formation has been considered a strategy to reduce the side-effects of the cisplatin therapy (27).

ER stress activates the UPR pathways through three major stress sensors: both α and β isoforms of IRE1, ATF6, and PERK (28). The activated pathways work in a concerted manner to arrest cell cycle and up-regulate ER stress-associated chaperone proteins through transcription of genes, such as ATF4 and CHOP. At basal conditions, PERK and IREα are bound to the ER chaperone GRP78 and remain in an inactive form (29). Under ER stress conditions, GRP78 preferentially binds to misfolded proteins, which releases PERK and IREα to activate the associated stress response. Homodimerization of IRE1 (both α and β isoforms) and activation of PERK via dissociation from the GRP78 binding are central to the ignition of the UPR response (30). These stress sensors induce the alarm stress pathways, including those driven by JNK and p38, through binding to adaptor proteins. Our findings indicated that cisplatin-induced ROS increased ER stress in TH-1 cells by the dissociation of GRP78 from PERK and IREα, and fucoidan inhibited cisplatin-induced ER stress by maintaining the complex of GRP78/PERK and GRP78/IREα. These results suggest that fucoidan protected TH-1 cells against cisplatin-mediated ER stress through stabilization of the complex of GRP78 with PERK and IREα.

Figure 6.
Figure 6.

Fucoidan-mediated expression of PrPC reversed cell-cycle arrest induced by cisplatin. (A) BrdU incorporation was measured using ELISA assay after treatment of fucoidan-pretreated TH-1 cells with si-PRNP. Values represent the mean±SEM. *p<0.05, and **p<0.01 vs. untreated TH-1 cells, ##p<0.01 vs. cisplatin treatment of TH-1 cells, $$p<0.01 vs. fucoidan-treated TH-1 cells pretreated with si-PRNP. (B) Expression of CDK2, cyclin E, CDK 4, and cyclin D1 was detected by western blotting after treatment of fucoidan-pretreated TH-1 cells with si-PRNP. (C) The expression levels were determined by densitometry relative to β-actin. Values represent the mean±SEM. **p<0.01 vs. untreated TH-1 cells, #p<0.05, and ##p<0.01 vs. cisplatin treatment of TH-1 cells, $$p<0.01 vs. fucoidan-treated TH-1 cells pretreated with si-PRNP.

Figure 7.
Figure 7.

Schematic representation of proposed mechanisms by which fucoidan protects ROS-induced apoptosis via up-regulation of PrPC -dependent antioxidant capacity. Cisplatin-induced ER stress and DNA damage lead to decreased cell survival and proliferation. Pretreatment of TH-1 cells with fucoidan reduced ER stress and DNA damage by increasing the expression of PrPC, which promotes activation of antioxidative enzyme.

PrPC has a protective role as it withstands various stress conditions such as hypoxia, ischemia, and exocytoxicity (31, 32). The anti-apoptotic and pro-survival activities of PrPC against oxidative stress have been shown to promote long-term neuroprotection and angiogenesis in the ischemic brain (19, 33). In support, reduced expression of PrPC and increased accumulation of ROS have been found in chronic kidney disease (CKD) patients (34). Our results showed that fucoidan increases the expression of PrPC and MnSOD, a type of SOD antioxidant enzyme. The up-regulation of PrPC augmented the antioxidative capacity of TH-1 cells by concomitantly enhancing SOD and catalase activities. A previous study has shown that fucoidan activates the antioxidative activities in mesenchymal stem cells (MSCs) under oxidative stress by up-regulating the expression of the major antioxidative enzyme MnSOD (35). Aligning with our rationale regarding the effects of ROS, cell cycle arrest and apoptotic signaling induced by cisplatin were recovered towards the homeostatic levels following the treatment with fucoidan. By silencing the expression of PrPC, however, the protective effects of the fucoidan were eradicated. These findings indicate that PrPC is a downstream effector of fucoidan-mediated function in up-regulating antioxidative enzymes.

Our results confirmed that accumulation of ROS induced DDR and the subsequent cell-cycle arrest and apoptosis when oxidative stress was not mitigated and fucoidan prevented excessive activation of DDR pathway through ER stress response. These findings indicated that fucoidan prevents the over-activation of DDR pathway in TH-1 cells in response to cisplatin through inhibition of ER stress, suggesting that fucoidan could be a protective agent to inhibit the side effects of cisplatin. Taken together, our findings indicated fucoidan as a potential drug that may inhibit cisplatin-related pathologies by suppressing the formation of ROS, inhibiting ER stress, DNA damage, and apoptosis and augmenting cell survival and proliferation (Figure 7). This study showed for the first time that PrPC-dependent antioxidant activity is the underlying mechanism of the fucoidan action. Therefore, we suggest fucoidan as a potential adjuvant drug for the patients who suffer from renal injury side effects of anticancer therapy.

Acknowledgements

This work was supported by the National Foundation grant funded by the Korean government (NRF-2016R1D-1A3B01007727). The funders had no role in study design, data collection or analysis, decision to publish, or in the preparation of the manuscript.

Footnotes

  • Authors' Contributions

    Hyung Joo Kim: study concept and design, acquisition of data, analysis and interpretation of data, and drafting of the manuscript. Yeo Min Yun: study concept and design, acquisition of data, analysis and interpretation of data, and drafting of the manuscript. Jun Hee Lee: study concept and design, analysis and interpretation of data, and drafting of the manuscript. Sang Hun Lee: study concept, data acquisition, analysis, and interpretation, drafting of the manuscript, procurement of funding, and study supervision.

  • Conflicts of Interest

    The Authors declare no conflicts of interest regarding this study. The funding body had no role in the study design, data collection or analysis, the decision to publish, or in the preparation of the manuscript.

  • Received September 4, 2019.
  • Revision received September 13, 2019.
  • Accepted September 16, 2019.

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Protective Role of Fucoidan on Cisplatin-mediated ER Stress in Renal Proximal Tubule Epithelial Cells
HYUNG JOO KIM, YEO MIN YOON, JUN HEE LEE, SANG HUN LEE
Anticancer Research Oct 2019, 39 (10) 5515-5524; DOI: 10.21873/anticanres.13744
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