YGYNO-975705; No. of pages: 10; 4C: Gynecologic Oncology xxx (2014) xxx–xxx

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Brown algae phlorotannins enhance the tumoricidal effect of cisplatin and ameliorate cisplatin nephrotoxicity Yeong-In Yang a,b, Ji-Hye Ahn a,b, Youn Seok Choi c, Jung-Hye Choi a,b,⁎ a b c

Department of Life & Nanopharmaceutical Science, Kyung Hee University, Seoul, South Korea Division of Molecular Biology, College of Pharmacy, Kyung Hee University, Seoul, South Korea Department of Obstetrics and Gynecology, School of Medicine, Catholic University of Daegu, Daegu, South Korea

H I G H L I G H T S • Phlorotannin-rich extract of Ecklonia cava (PREC) enhanced the tumor growth-inhibitory effect of cisplatin and ameliorated cisplatin-induced nephrotoxicity in vivo. • PREC enhanced ovarian cancer cell apoptosis by cisplatin via the ROS/Akt/NFκB pathway and suppressed cisplatin-induced normal kidney cell damage. • Dieckol, a major phlorotannin of PREC, significantly enhanced the tumoricidal effect of cisplatin in vivo.

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Article history: Received 18 August 2014 Accepted 15 November 2014 Available online xxxx Keywords: Ecklonia cava Phlorotannins Cisplatin Ovarian cancer ROS/Akt/NFκB pathway Nephrotoxicity

a b s t r a c t Objective. The clinical application of cisplatin is limited due to its drug resistance and side effects. We investigated the effect of a phlorotannin-rich extract from the edible brown alga Ecklonia cava (PREC) and its major phlorotannin (dieckol) on cisplatin responsiveness and side effects. Methods. The A2780 and SKOV3 ovarian cancer cell lines and the SKOV3-bearing mouse model were used. The MTT assay was applied to assess cell viability, and the annexin V assay was employed for apoptosis analysis. Reactive oxygen species (ROS) production and protein expression were assessed by H2DCFDA staining and Western blotting, respectively. Results. We found that PREC enhanced the tumor growth-inhibitory effect of cisplatin and diminished cisplatin-induced nephrotoxicity and weight loss in SKOV3-bearing mice. PREC augmented cisplatininduced apoptosis by activating caspases in SKOV3 and A2780 ovarian cancer cells. In addition, a combination of PREC and cisplatin-induced ovarian cancer cell apoptosis by downregulating the Akt and NFκB pathways. We further demonstrated that PREC increased intracellular ROS and that antioxidants significantly attenuated Akt-NFκB activation and apoptosis in ovarian cancer cells. In contrast, PREC inhibited cisplatin-induced ROS production and cell death in normal HEK293 kidney cells. Dieckol, a major compound in PREC, significantly enhanced the inhibition of tumor growth by cisplatin with less weight loss and kidney damage in a mouse model. Conclusion. These data suggest that brown algae phlorotannins may improve the efficacy of platinum drugs for ovarian cancer by enhancing cancer cell apoptosis via the ROS/Akt/NFκB pathway and reduce nephrotoxicity by protecting against normal kidney cell damage. © 2014 Elsevier Inc. All rights reserved.

Introduction Ovarian cancer is the most lethal gynecological malignancy, as most patients with ovarian cancer are diagnosed at late stages due to a lack of effective screening strategies and specific symptoms associated with ⁎ Corresponding author at: Division of Molecular Biology, College of Pharmacy, Kyung Hee University, Dongdaemun-Gu, Hoegi-Dong, Seoul, 130-701, South Korea. Fax: +82 2 962 0860. E-mail address: [email protected] (J.-H. Choi).

early stage disease [1]. Despite the great achievements made over the past three decades in cytoreductive surgery and combination chemotherapy, ovarian cancer remains a significant threat to women internationally [2]. Cisplatin, or cis-diamminedichloroplatinum, has been used to treat a variety of malignancies including ovarian cancer. However, the administration of cisplatin is often associated with serious side effects including nephrotoxic and neurotoxic events. The accompanying toxicity to normal tissue and the acquisition of drug resistance poses major problems during cisplatin therapy [3,4]. The combined use of

http://dx.doi.org/10.1016/j.ygyno.2014.11.015 0090-8258/© 2014 Elsevier Inc. All rights reserved.

Please cite this article as: Yang Y-I, et al, Brown algae phlorotannins enhance the tumoricidal effect of cisplatin and ameliorate cisplatin nephrotoxicity, Gynecol Oncol (2014), http://dx.doi.org/10.1016/j.ygyno.2014.11.015

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two or more chemotherapy agents is often advantageous, as it may permit lowering drug dosages and consequently decreasing cytotoxicity, which reduces the opportunity for the development of drug resistance by cancer cells [5]. Therefore, identifying agents that can sensitize tumor cells to cisplatin with no or less toxicity to normal tissue would have an important impact on cisplatin-based therapy. Many studies have demonstrated the anticancer effect of natural materials from terrestrial and marine resources. Marine-derived natural products are known for their huge diversity of chemical structures, and their unique structures are frequently associated with special mechanisms of action by which they may elicit unexpected biological activities [6]. Marine algae are good candidates for these unique biological activities due to their diverse classes of active secondary metabolites. Polyphenols are one of the most common classes of secondary metabolites in marine plants. The unique polyphenolic compounds phlorotannins, which are formed by the polymerization of phloroglucinol (1,3,5tryhydroxybenzene) units, have been identified in Ecklonia sp. [7]. Phlorotannins such as eckol (a closed-chain trimer of phloroglucinol), 6,6′-bieckol (a hexamer), dieckol (a hexamer), and phlorofucofuroeckol (a pentamer) have been identified in the edible brown algae Ecklonia cava (E. cava) and have been extensively studied for their potential health benefits. For example, the promising effects of E. cava against radical-mediated oxidative stress [8], photodamage [9], allergy [10], diabetes [11], inflammation [12], and viral and microbial infections [13] have been reported. Phlorotannins are believed to be promising as nutraceuticals and pharmaceuticals due to their vast range of biological activities. Thus, a better understanding of the molecular mechanisms behind the modulation of cellular responses is needed to develop and optimize new therapeutic strategies using E. cava. In the present study, we investigate the potential effect of the phlorotannin-rich extract of the edible brown alga E. cava (PREC) to enhance cisplatin cytotoxicity and reduce kidney damage in ovarian cancer in vivo and in vitro. We also examined the reactive oxygen species (ROS)-mediated mechanisms of action of the combination of cisplatin and PREC. In addition, we studied the role of dieckol, a major phlorotannin in PREC, for PREC-induced anticancer effects.

Materials and methods Materials PREC and dieckol (N99% purity) used for this study was kindly supplied by Livechem, Inc. (Daejeon, South Korea). The method to prepare the phlorotannins-rich extract of E. cava (PREC) has been previously described [14]. The total polyphenol content of PREC as phloroglucinol equivalent was about 98%, and major phlorotannins in the PREC were as follows: 2-O-(2,4,6-trihydroxyphenyl)-6,6′-bieckol, 6,6′-bieckol, 8,8′-bieckol, 7-phloroeckol, eckol, phlorofurofukoeckol, and dieckol (16.6%), as determined by HPLC [Waters, column:Spherisorb S10ODS2 column (20 × 250 mm2); eluent: 30% aqueous EtOH; flow rate: 3.5 ml/min]. RPMI 1640 medium, fetal bovine serum (FBS), penicillin, and streptomycin were obtained from Life Technologies Inc. (NY, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), RNase A, leupeptin, aprotinin, phenylmethylsulfonyl fluoride (PMSF), Triton X-100, and propidium iodide (PI) were purchased from Sigma Chemical Co. (MO, USA). Antibodies against p65, p50, Bcl2, BclxL, FLIP/L, Akt, proliferating cell nuclear antigen (PCNA), and β-actin were purchased from Santa Cruz Biotechnology (CA, USA). Antibodies against X-linked inhibitor of apoptosis protein (XIAP), caspase-3, and caspase-8 were purchased from BD Biosciences, Pharmingen (CA, USA). Antibodies against caspase-9 and phospho-Akt were purchased from Cell Signaling Technology (MA, USA). pcDNA3-Akt-myr, pcDNA3-Akt-DN, and pCMV4-p65 were obtained from Addgene (MA, USA). z-VAD-fmk, z-DEVD-fmk, and z-LEHD-fmk were obtained from Calbiochem (CA, USA).

Cell cultures and MTT assay Ovarian cancer SKOV3 and A2780 cells were obtained from American Type Culture Collection. Cells were cultured in RPMI 1640 supplemented with 5% fetal bovine serum, penicillin (100 U/ml) and streptomycin sulfate (100 μg/ml) (Life Technologies, NY, USA) in a humidified atmosphere of 5% CO2 95% air at 37 °C. Cell viability was assessed using an MTT assay. Briefly, the cells (5 × 104) were seeded in each well containing 50 μl of RPMI medium in a 96-well plate. After 24 h, cisplatin and/or PREC were added. After 48 h, 50 μl of MTT solution (5 mg/ml in PBS) was added to the medium, and the cells were incubated at 37 °C for 4 h. The optical density was measured at 540 nm using a microplate spectrophotometer (SpectraMax; Molecular Devices, CA, USA) to determine the cell viability. Propidium iodide (PI) staining and annexin V and PI double staining Cells were fixed and permeabilized with 70% ice-cold ethanol at 4 °C for 1 h. Cells were washed once with PBS and resuspended in a staining solution containing PI (50 μg/ml) and RNase A (250 μg/ml). The mixture was incubated for 30 min at room temperature in a dark place and analyzed by FC500 fluorescence-activated cell sorting (FACS) cater-plus flow cytometry (Beckman Coulter, CA, USA). A total of 10,000 events were acquired for analysis using Cell Quest software. For annexin V and PI double staining, cells were suspended with 100 μl of binding buffer and stained with 5 μl of FITC-conjugated annexin V and 5 μl of PI (50 μg/ml). The mixture was incubated for 15 min at room temperature in a dark place and analyzed by FACS. Western blot analysis Protein samples of cell lysate were mixed with an equal volume of 5 × SDS sample buffer, boiled for 4 min, and then separated on 10–12% SDS–PAGE gels. After electrophoresis, proteins were transferred to polyvinylidene difluoride membranes. The membranes were blocked in 5% non-fat dry milk for 1 h, washed, and incubated with specific antibodies in Tris-buffered saline (TBS) containing Tween-20 (0.1%) overnight at 4 °C. Primary antibodies were removed by washing the membranes three times in TBS-T, and then the membranes were incubated for 1 h with horseradish peroxidase-conjugated secondary antibody for 1 h, and visualized using the ECL chemiluminescent system (Amersham Pharmacia Biotech, ON, Canada). Following three washes in TBS-T, immuno-positive bands were visualized by enhanced chemiluminescence and exposed to Image Quant LAS-4000 (Fujifilm Life. Sciences, Tokyo, Japan). Measurement of reactive oxygen species (ROS) The intracellular accumulation of ROS was determined using the fluorescent probe H2DCFDA. H2DCFDA was commonly used to measure H2O2. Cells were collected by centrifugation 30 min before treatment with the cytotoxic agents, resuspended in PBS, and loaded with 20 μM H2DCFDA. The fluorescence was measured at the desired time intervals by flow cytometry. Animal study BALB/c athymic female nude mice (n = 20) weighing 20–25 g from NARA Biotech (Seoul, Korea) were used for the studies. Ovarian carcinoma was created by subcutaneously (s.c.) inoculating 5 × 106 SKOV3 cells (100 μl) into the flank of each mouse and tumors were allowed to grow for 1 week. The tumor-bearing mice were randomly divided into 5 or 6 groups (5 mice/group). For 4 consecutive weeks, PREC (75 and 150 mg/kg) or dieckol (50 and 100 mg/kg) was given orally three times per week while cisplatin was administered via intraperitoneal (i.p.) injection at doses of 3 mg/kg/day three times per week. The

Please cite this article as: Yang Y-I, et al, Brown algae phlorotannins enhance the tumoricidal effect of cisplatin and ameliorate cisplatin nephrotoxicity, Gynecol Oncol (2014), http://dx.doi.org/10.1016/j.ygyno.2014.11.015

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tumor size was measured using calipers and the tumor volume was estimated by the formula: tumor volume (mm3) = 1/2 (L × W2), where L is the length and W is the width of the tumor. On day 28, the mice were sacrificed and the tumors were excised from the body for measuring the tumor weight. Additionally, blood was drawn from the inferior vena cava into a heparin-coated tube, and the serum was obtained by centrifuging the blood at 15,000 g for 15 min at 4 °C. Serum creatinine, blood urea nitrogen (BUN), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were measured using colorimetric assay kit (Asan Pharmaceutical, Seoul, South Korea) according to the manufacturer's instructions. The serum creatinine and BUN levels were measured as indicators of kidney function while the ALT and AST levels were measured to evaluate the liver function.

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mouse model. As shown in Fig. 1A and B, PREC (150 mg/kg) alone did not show a significant inhibitory effect on tumor growth. However, adding PREC (150 mg/kg) markedly increased the potency of cisplatin (3 mg/kg) in the SKOV3 xenograft mice model. In addition, PREC (150 mg/kg) combined with cisplatin (3 mg/kg) reversed the weight loss caused by cisplatin (Fig. 1C). Next, the protective effects of PREC against cisplatin-induced hepatotoxicity and nephrotoxicity were evaluated. Liver toxicity was not observed in any of the mice treated with cisplatin, PREC, or their combination (Supplementary Fig. 1S). However, cisplatin (3 mg/kg) markedly increased both blood urea nitrogen (BUN) and creatinine levels, whereas concurrent administration of PREC (150 mg/kg) with cisplatin significantly prevented the rise in BUN and creatinine, suggesting a potential nephroprotective effect of PREC (Fig. 1D).

Statistical analysis Results are expressed as the mean ± SD of triplicate experiments. Statistically significant values were compared using analysis of Student's t-test and one-way ANOVA, and *P-values of b0.05 were considered statistically significant. Results PREC enhanced the inhibition of tumor growth and nephrotoxicity induced by cisplatin in an ovarian cancer xenograft mouse model We first investigated the effect of PREC, cisplatin, and their combination on tumor growth and tissue damage in an ovarian cancer xenograft

Combined treatment with PREC and cisplatin-induced apoptosis by activating caspases in ovarian cancer cells Due to our promising in vivo results, we investigated the effect of PREC and its combination with cisplatin on cell growth in ovarian cancer cells. PREC alone showed only a mild cell growth-inhibitory effect in SKOV3 and A2780 ovarian cancer cells (IC50 = 131.30 and 80.01 μg/ml, respectively) (Supplementary Table S1). However, treatment with cisplatin (1, 3, or 5 μM) in combination with PREC (35, 50, 75, or 100 μg/ml) for 48 h markedly enhanced cisplatin-induced cell death as shown by the sub G1 population in both SKOV3 and A2780 cells (Fig. 2A and B).

Fig. 1. PREC enhances tumor growth-inhibitory effect of cisplatin and reduced cisplatin-induced nephrotoxicity in SKOV3-bearing xenograft models. SKOV3 xenografts were treated with vehicle, cisplatin (3 mg/kg) or PREC (75 and 150 mg/kg) three times per week for 4 weeks. When treatment was finished, the mice were sacrificed and tumor volume (mm3) (A) and tumor weights (g) (B) were measured. (C) Mice body weight changes were measured two times per week during the treatment period. (D) Plasma creatinine and BUN levels were measured using a colorimeter testing kit. #P b 0.05 vs. the control group (vehicle-treated group); *P b 0.05 vs. cisplatin-treated group.

Please cite this article as: Yang Y-I, et al, Brown algae phlorotannins enhance the tumoricidal effect of cisplatin and ameliorate cisplatin nephrotoxicity, Gynecol Oncol (2014), http://dx.doi.org/10.1016/j.ygyno.2014.11.015

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Please cite this article as: Yang Y-I, et al, Brown algae phlorotannins enhance the tumoricidal effect of cisplatin and ameliorate cisplatin nephrotoxicity, Gynecol Oncol (2014), http://dx.doi.org/10.1016/j.ygyno.2014.11.015

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The annexin V–FITC staining assay was performed to further determine whether the growth-inhibitory effect was associated with the induction of apoptosis. As shown in Fig. 2C, treatment with the combination of PREC (50 μg/ml) and cisplatin (5 μM) for 24 h significantly increased the populations of annexin V-positive cells (apoptotic cells) in SKOV3 cells. To confirm the apoptotic effect of the combination of the two agents, activation of caspases, important mediators of apoptosis, was investigated using Western blot analysis. The PREC and cisplatin combination (PREC 50 μg/ml and cisplatin 5 μM) significantly activated caspase-9, caspase-8, and caspase-3, whereas treatment with PREC or cisplatin alone led to mild activation of the caspases (Fig. 2D) in SKOV3 cells. To confirm the involvement of caspases in PREC and cisplatin-induced apoptosis, we used various caspase inhibitors such as z-VAD-fmk (a broad caspase inhibitor), z-DEVD-fmk (a specific caspase-3 inhibitor), and z-LEHD-fmk (a specific caspase-9 inhibitor) in SKOV3 cells. All of these inhibitors markedly attenuated PREC and cisplatin-induced cell death, as shown by the MTT assay (Fig. 2E), indicating that caspases play an essential role in PREC and cisplatin-induced apoptosis in ovarian cancer cells.

Combined treatment with PREC and cisplatin-induced apoptosis by inhibiting the NFκB and Akt signaling pathways in ovarian cancer cells The NFκB pathway regulates the expression of several genes involved in cell survival or anti-apoptosis. Considering that various polyphenolic compounds enhance apoptosis by suppressing the NFκB pathway [15,16], we investigated the potential attenuation of this pathway by PREC and cisplatin treatment in SKOV3 cells. As shown in Fig. 3A, treatment with PREC (50 and 100 μg/ml) and cisplatin (5 μM) for 48 h significantly suppressed nuclear translocation of NFκB compared with that in untreated controls or those treated with only one of the agents. The expression levels of the NFκBdependent anti-apoptotic genes (Bcl2, BclxL, FLIP/L, and XIAP) were elucidated. Western blot analysis revealed that the combined treatment significantly decreased protein expression of those genes (Fig. 3A). In addition, overexpression of p65 significantly inhibited cell death induced by the combination as well as NFκB-dependent genes and caspase activation (Fig. 3B and C). Because several studies have suggested that Akt is an upstream regulator of NFκB signaling [15,17], we investigated involvement of the PI3K/Akt pathway in PREC and cisplatin-induced apoptosis in SKOV3 cells. We found that PREC (50 μg/ml) and cisplatin (5 μM) markedly inhibited activation of Akt in a time-dependent manner (Fig. 4A). In addition, inhibiting Akt with the dominant-negative form of Akt vector (Akt-DN) transfection resulted in enhanced cell death in PREC and cisplatin combined treated cells (Fig. 4B). In contrast, the constitutively active form of Akt (Akt-myr) significantly inhibited cell death induced by the combination (PREC 50 μg/ml and cisplatin 5 μM). Furthermore, when Akt was activated by Akt-myr overexpression, cisplatin-PREC-induced inhibition of NFκB translocation, NFκBdependent gene expression, and caspase-3 activation were markedly attenuated (Fig. 4C). Notably, the inhibition of Akt remained unchanged when p65 was overexpressed (Supplementary Fig. S2), suggesting that Akt may act as an upstream factor of the NFκB pathway. Taken together, these data suggest that PREC may enhance the cisplatin effect by inhibiting Akt and NFκB activation and cause a marked

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decrease in anti-apoptotic genes, shifting the balance toward cell death and restoring the cisplatin-induced apoptotic response in ovarian cancer cells.

Combined treatment with PREC and cisplatin-induced apoptosis by inducing ROS in ovarian cancer cells ROS production has been recognized as an early signal that mediates apoptotic cell death [18]. Thus, we next examined whether PREC and cisplatin-induced anticancer activities were associated with the generation of ROS. As shown in Fig. 5A, PREC (50 and 100 μg/ml) or cisplatin (5 μM) treatment alone induced ROS production, and the combined treatment markedly increased ROS levels in SKOV3 cells. In the presence of the antioxidant N-acetyl-L-cysteine (NAC), the combined treatment reduced ROS production (Fig. 5A) and significantly suppressed apoptosis (Fig. 5B). In addition, treatment with NAC markedly attenuated PREC and cisplatin-induced phosphorylation of Akt, translocation of NFκB, expression of NFκB-dependent anti-apoptotic genes, and activation of caspase-3 (Fig. 5C). These data identify ROS as the upstream regulatory factor for PREC augmentation of cisplatin-induced apoptosis in ovarian cancer.

Combined treatment with PREC and cisplatin ameliorated cisplatin-induced cytotoxicity in human normal kidney cells Cisplatin-induced nephrotoxicity has been suggested to be associated with kidney cell damage by ROS [19]. Thus, we investigated whether PREC protects against cisplatin-induced normal kidney cell death by modulating ROS production. As shown in Fig. 5D, cisplatin-induced death of normal HEK293 kidney cells was significantly reversed by PREC treatment (50 μg/ml). Similar to cancer cells, cisplatin treatment (5 μM) markedly increased intracellular ROS levels in HEK293 cells (Fig. 5E). In contrast, PREC (50 and 100 μg/ml) reduced endogenous ROS levels in HEK293 cells, and the combination of PREC and cisplatin maintained ROS at relatively low levels. These data suggest the PREC may protect against cisplatin-induced nephrotoxicity by reducing ROS stress in normal kidney cells.

Dieckol isolated from E. cava enhanced anticancer activities by cisplatin in an ovarian cancer xenograft mouse model Dieckol, a major component of PREC, has been demonstrated to exert several pharmacological activities including anti-metastatic activity [20–24]. We further investigated whether dieckol is associated with the PREC-enhanced cisplatin responsiveness in a xenograft mouse model. Dieckol (50 or 100 mg/kg) alone showed a mild inhibitory effect on tumor growth (Fig. 6A and B). In addition, it is of note that dieckol in combination with cisplatin (3 mg/kg) produced a significant reduction in tumor growth compared with that of dieckol alone, cisplatin alone, and the control. Mice in the control and cisplatin alone groups lost weight during the treatment, whereas those administered the combined treatment maintained their body weight (Fig. 6C). Dieckol slightly suppressed cisplatin-increased creatinine and BUN levels, but the suppression did not reach statistical significance (P N 0.05; Fig. 6D).

Fig. 2. PREC enhances cisplatin-induced caspase-dependent apoptosis in ovarian cancer cells. (A) Human ovarian cancer cells (SKOV3 and A2780) were treated with cisplatin (1, 3, or 5 μM) and PREC (35, 50, 75, or 100 μg/ml) for 48 h and then stained with PI. (B) The percentage of cells with sub G1 DNA content was taken as a measure of the cell death. The data shown represent means ± SD of three independent experiments. ⁎P b 0.05 vs. the control group. (C) SKOV3 cells were treated with cisplatin (5 μM) and PREC (50 μg/ml) for 24 h and co-stained with FITC-conjugated annexin V and PI for flow cytometric analysis. Representative histograms indicate the percentages of annexin V and PI positive cells. ⁎P b 0.05 vs. the control group. (D) SKOV3 cells were treated cisplatin (5 μM) and PREC (50 and 100 μg/ml) and then harvested after 48 h for Western blot analysis. (E) MTT assays were performed using the broad-spectrum inhibitor z-VAD-fmk (50 μM), caspase-3 specific inhibitor z-DEVD-fmk (50 μM), and caspase-9 specific inhibitor z-LEHD-fmk (50 μM). SKOV3 cells were pretreated with and without caspase inhibitor for 30 min, treated with cisplatin (5 μM) and PREC (50 μg/ml) for 24 h. #P b 0.05 vs. the control group; *P b 0.05 vs. combination-treated group.

Please cite this article as: Yang Y-I, et al, Brown algae phlorotannins enhance the tumoricidal effect of cisplatin and ameliorate cisplatin nephrotoxicity, Gynecol Oncol (2014), http://dx.doi.org/10.1016/j.ygyno.2014.11.015

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Fig. 3. NFκB signaling pathway is inhibited by the PREC–cisplatin combination in ovarian cancer cells. (A) SKOV3 cells were treated cisplatin (5 μM) and PREC (50 and 100 μg/ml) and then harvested after 48 h treatment. Nuclear extracts (N) were then prepared for Western blot with a specific p50 and p65 NFκB antibody, as described in the Materials and methods section. Proliferating cell nuclear antigen (PCNA) was used as the internal control for the nuclear fraction. (B and C) SKOV3 cells were transfected with pCMV p65 (1 and 2 μg/ml) or the empty vector. Transfected cells were treated with cisplatin (5 μM) and PREC (50 μg/ml) and then analyzed after 24 h treatment. (B) MTT assays were performed to examine the cell viability. # P b 0.05 vs. the control group; *P b 0.05 vs. combination-treated group. (C) The expression levels of NFκB-dependent genes and pro-caspase-3 were analyzed by Western blot.

Discussion Platinum-based drugs have been used as first-line chemotherapeutic agents for treating a variety of solid tumors, including ovarian cancer. However, their clinical application is limited due to drug resistance and side effects. Therefore, current efforts are being directed toward discovering novel approaches to overcome drug resistance and reduce side effects. Several studies have attempted to identify proper natural products for combined therapy. Many recent studies have demonstrated E. cava extract and several phlorotannins isolated from the brown algae have various anticancer activities such as anti-metastasis [22–24], anti-angiogenesis [25], and anti-proliferation [26]. For example, phloroglucinol isolated from E. cava was suggested to inhibit the bioactivities of endothelial progenitor cells, suppressing tumor angiogenesis [25]. Dieckol inhibited the invasion and migration of HT1080 human fibrosarcoma and B16 mouse melanoma cells [22–24]. Dioxinodehydroeckol induced apoptosis in human breast cancer [26]. Based on the potential anticancer activities of the brown algae phlorotannins, in the present study, we evaluated the anticancer efficacy of a phlorotannin-rich extract from E. cava (PREC) and its combined effect with cisplatin in an in vivo preclinical ovarian cancer model. We found that the E cava extract alone had a mild inhibitory effect on

tumor growth and significantly enhanced the tumoricidal effect of cisplatin in SKOV3-bearing mice. Unexpectedly, PREC significantly decreased serum BUN and creatinine levels elevated by cisplatin, suggesting that PREC ameliorates cisplatin-induced nephrotoxicity. In addition, cisplatin-induced weight loss was prevented by administering PREC. Moreover, the change in body weight was correlated with food intake during the 28 days (Supplementary Fig. S3). These results suggest that the combined therapy of cisplatin and PREC may be helpful not only in tumor growth control but also in improving cisplatininduced kidney toxicity and loss of appetite and weight during ovarian cancer treatment. We further demonstrated that PREC augmented cisplatin-induced apoptosis by activating caspases in ovarian cancer cells, suggesting that the inhibitory effect of the combination on tumor growth in mice is associated with the induction of apoptotic cell death in cancer cells. Further studies are needed to investigate if PREC and its combination with cisplatin also have inhibitory effect on migration, invasion, and angiogenesis in ovarian cancer mice model. The present study represents the first in vivo experimental investigation to show the adjuvant potential of E. cava phlorotannins during cisplatin therapy for ovarian cancer. Various polyphenolic compounds seem to enhance apoptosis by suppressing the NFκB pathway. For example, tectorigenin, a flavonoid

Please cite this article as: Yang Y-I, et al, Brown algae phlorotannins enhance the tumoricidal effect of cisplatin and ameliorate cisplatin nephrotoxicity, Gynecol Oncol (2014), http://dx.doi.org/10.1016/j.ygyno.2014.11.015

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Fig. 4. Akt/PI3K signaling pathway is inhibited by the PREC–cisplatin combination in ovarian cancer cells. (A) SKOV3 cells were treated with cisplatin (5 μM) and PREC (50 μg/ml) for the indicated times (0, 1, 3, and 6 h) and then harvested for Western blot analysis. (B and C) SKOV3 cells were transfected with Akt-myr vector, Akt-DN vector, or the empty vector. (B) MTT assays were performed to examine the cell viability. #P b 0.05 vs. the control group; *P b 0.05 vs. combination-treated group. (C) Nuclear extracts (N) were then prepared for Western blotting with a specific p50 and p65 NFκB antibody. The expression levels of NFκB-dependent genes and pro-caspase-3 were analyzed using specific antibodies.

phenolic compound, sensitizes paclitaxel-resistant human ovarian cancer cells by inactivating the Akt/IKK/IκB/NFκB signaling pathway [15]. Anto et al. [16] showed that overexpression of p65 in mouse fibrosarcoma cells causes resistance to polyphenolic curcumin-induced apoptosis. In addition to the NFkB pathway, the PI3K/Akt pathway has been suggested to be involved in the chemosensitizing effect of polyphenols. For example, the citrus flavonoid tangeretin sensitizes cisplatinresistant human ovarian cancer cells by downregulating the PI3K/Akt pathway [27]. In the present study, we found that the PREC–cisplatin combination significantly inhibited activation of the NFκB and the inhibitory effect is at least in part mediated by inactivating Akt in ovarian cancer cells. These results agree with the findings of other studies, in which activation of PI3K/Akt promoted activation of the NFκB pathway [15,17]. As cancer cells show higher levels of endogenous ROS compared with those in normal cells, a toxic threshold can be easily achieved in cancer cells [28]. Considering that only 5–10% of covalently bound, cell-associated cisplatin is found in the DNA fraction [29], cisplatin has been suggested to induce cell death independent of DNA damage, and one of the recognized mechanisms for this induction is the formation of cisplatin-induced ROS [30]. Berndtsson et al., using the HCT116 colon carcinoma cell line and a melanoma cell line, found that low doses of cisplatin induce DNA damage, whereas high doses induce ROS, ultimately leading to apoptosis [31]. Furthermore, they found that cell death could be inhibited by adding superoxide scavengers and that cell death was DNA damage independent [29]. In addition to chemotherapeutic agents, many naturally occurring compounds exert anticancer effects by inducing ROS [32,33]. Here, we demonstrated that PREC-induced ROS production acted upstream of apoptosis via the Akt/NFκB pathway in ovarian cancer cells. ROS formation can activate the Akt pathway, which is upstream of the NFκB pathway, and can therefore induce activation of NFκB [34]. Notably, many sea and

terrestrial polyphenols are generally recognized as antioxidants and show cytoprotective effects in some cells by reducing ROS levels [20, 35–37]. These observations suggest that polyphenols can act as a double-edged sword, both as an antioxidant and a prooxidant. For example, several flavonoids, which are well-known antioxidant scavengers, generate ROS such as hydrogen peroxide to induce apoptosis and cell death in cancer cell lines [38]. Brown algal phlorotannins have potent antioxidant activities [39]. In this study, we found that PREC inhibited cisplatin-induced ROS production and cell death in normal HEK293 kidney cells, whereas it acted as an oxidant in ovarian cancer cells. The possibility exists that some compounds in PREC mainly induced ROS production in cancer cells, whereas others reduced ROS in normal kidney cells. The molecular mechanism underlying the dual action of E. cava polyphenols remains to be investigated. Oxidative stress is generally believed to underlie the pathogenesis of cisplatin-induced nephrotoxicity [40,41]. In fact, cisplatin induces free radicals and apoptosis of renal tubules [42], the latter of which is characterized by changes in BUN, creatinine, and glutathione (GSH) levels, as well as lipid peroxidation. A study suggested that upregulation of ROS is associated with an impaired glomerular filtration rate [43] and antioxidants maintain intracellular reduced GSH concentrations, restore cellular defense mechanisms, and block lipid peroxidation, protecting renal cells against the noxious effects of a wide variety of nephrotoxic agents [44,45]. These data suggest that the antioxidant activities of PREC in normal kidney cells shown here may play a key role in the nephroprotective effect of PREC to cisplatin treatment as shown by reduced BUN and creatinine levels in the in vivo study. Further mechanistic studies are needed to fully understand the nephroprotective effect of PREC. Dieckol, a trimeric compound of phloroglucinol with a dibenzeno1,4-dioxin skeleton, is the major phlorotannin in PREC. Dieckol inhibits migration and invasion of B16 mouse melanoma and HT1080 human

Please cite this article as: Yang Y-I, et al, Brown algae phlorotannins enhance the tumoricidal effect of cisplatin and ameliorate cisplatin nephrotoxicity, Gynecol Oncol (2014), http://dx.doi.org/10.1016/j.ygyno.2014.11.015

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Fig. 5. PREC enhances cisplatin-induced ROS accumulation in ovarian cancer cells and reduces cisplatin-induced ROS accumulation in normal kidney cells. (A) SKOV3 cells were treated with PREC (50 and 100 μg/ml) for 30 min in the presence of NAC (5 mM) and then stained with H2DCFDA. (B) SKOV3 cells were treated with cisplatin (5 μM) and PREC (50 μg/ml) in the presence of NAC for 24 h, and then MTT and annexin V staining assay were performed. *P b 0.05 vs. vehicle-treated control group. (C) SKOV3 cells were pretreated with and without NAC (5 mM) for 1 h, treated with cisplatin (5 μM) and PREC (50 μg/ml) for 48 h. Western blot analysis was performed. (D) HEK293 normal kidney cells were treated cisplatin (5 μM) and PREC (50 μg/ml) for 48 h. Cytotoxicity was measured by MTT assay. #P b 0.05 vs. the control group; *P b 0.05 vs. combination-treated control group. (E) HEK293 cells were treated with cisplatin (5 μM) and PREC (50 or 100 μg/ml) for 30 min, and then stained with H2DCFDA.

fibrosarcoma cells [23,24,46]. However, the effects of dieckol on cancer cell growth and/or chemosensitivity are poorly understood. In this study, dieckol significantly enhanced the cisplatin response in SKOV3bearing mice. However, only a mild effect of dieckol on cisplatininduced BUN and creatinine levels was observed. These data suggest that the PREC-enhanced cisplatin responsiveness is likely due to the activities of the major phlorotannin dieckol and that dieckol may play some partial role in the nephroprotective activities. The identities of other compounds in PREC that play a key role in ameliorating cisplatin-induced kidney toxicity remain to be determined. Despite the accumulating evidence documenting the potential health benefits of brown algae phlorotannins, to the best of our knowledge, there has been no published study on the pharmacokinetics and bioavailability of the marine polyphenol phlorotannins [47]. The pharmacokinetics and bioavailability of terrestrial polyphenols such as flavonoids and catechins have been studied in human as well as animal models [48–52]. It has been suggested that most classes of polyphenols

are sufficiently absorbed to exert their biological activities [51]. However, considering the pharmacokinetics and bioavailability of polyphenols varies widely from one compound to another, further studies are still required to elucidate the exact pharmacokinetics and bioavailability of the phlorotannins. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.ygyno.2014.11.015. Conflict of interest statement The authors have no conflicts of interest to declare.

Acknowledgments This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant (to J.-H. Choi) funded by the Korea government (MEST; 2012R1A1A2039648 and NRF-2013R1A2A2A01067888).

Please cite this article as: Yang Y-I, et al, Brown algae phlorotannins enhance the tumoricidal effect of cisplatin and ameliorate cisplatin nephrotoxicity, Gynecol Oncol (2014), http://dx.doi.org/10.1016/j.ygyno.2014.11.015

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Fig. 6. Dieckol enhances tumor growth-inhibitory effect of cisplatin in SKOV3-bearing xenograft models. SKOV3 cells were inoculated subcutaneously into BALB/c nude mice, and SKOV3 xenografts were treated with vehicle, cisplatin (3 mg/kg) or dieckol (50 and 100 mg/kg) three times per week for 4 weeks. When treatment was finished, the mice were sacrificed and tumor volume (mm3) (A) and tumor weights (g) (B) were measured. (C) Mice body weight changes were measured two times per week during the treatment period. (D) Plasma creatinine and BUN levels were measured using a colorimeter testing kit. #P b 0.05 vs. the control group (vehicle-treated group); *P b 0.05 vs. cisplatin- treated group.

References [1] Pliarchopoulou K, Pectasides D. Epithelial ovarian cancer: focus on targeted therapy. Crit Rev Oncol Hematol 2011;79:17–23. [2] Harries M, Gore M. Part I: chemotherapy for epithelial ovarian cancer-treatment at first diagnosis. Lancet Oncol 2002;3:529–36. [3] Launay-Vacher V, Rey JB, Isnard-Bagnis C, Deray G, Daouphars M. Prevention of cisplatin nephrotoxicity: state of the art and recommendations from the European Society of Clinical Pharmacy Special Interest Group on Cancer Care. Cancer Chemother Pharmacol 2008;61:903–9. [4] Stewart DJ. Mechanisms of resistance to cisplatin and carboplatin. Crit Rev Oncol Hematol 2007;63:12–31. [5] Umanzor J, Aguiluz M, Pineda C, Andrade S, Erazo M, Flores C, et al. Concurrent cisplatin/gemcitabine chemotherapy along with radiotherapy in locally advanced cervical carcinoma: a phase II trial. Gynecol Oncol 2006;100:70–5. [6] Sithranga Boopathy N, Kathiresan K. Anticancer drugs from marine flora: an overview. J Oncol 2010;2010:214186. [7] Pal Singh I, Bharate SB. Phloroglucinol compounds of natural origin. Nat Prod Rep 2006;23:558–91. [8] Kang MC, Cha SH, Wijesinghe WA, Kang SM, Lee SH, Kim EA, et al. Protective effect of marine algae phlorotannins against AAPH-induced oxidative stress in zebrafish embryo. Food Chem 2013;138:950–5. [9] Cha SH, Ko CI, Kim D, Jeon YJ. Protective effects of phlorotannins against ultraviolet B radiation in zebrafish (Danio rerio). Vet Dermatol 2012;23:51–6 [e12]. [10] Li Y, Lee SH, Le QT, Kim MM, Kim SK. Anti-allergic effects of phlorotannins on histamine release via binding inhibition between IgE and Fc epsilonRI. J Agric Food Chem 2008;56:12073–80. [11] Lee SH, Jeon YJ. Anti-diabetic effects of brown algae derived phlorotannins, marine polyphenols through diverse mechanisms. Fitoterapia 2013;86:129–36. [12] Wijesinghe WAJP, Ahn G, Lee WW, Kang MC, Kim EA, Jeon YJ. Anti-inflammatory activity of phlorotannin-rich fermented Ecklonia cava processing by-product extract in lipopolysaccharide-stimulated RAW 264.7 macrophages. J Appl Phycol 2013;25: 1207–13. [13] Eom SH, Kim YM, Kim SK. Antimicrobial effect of phlorotannins from marine brown algae. Food Chem Toxicol 2012;50:3251–5. [14] Shin HC, Hwang HJ, Kang KJ, Lee BH. An antioxidative and antiinflammatory agent for potential treatment of osteoarthritis from Ecklonia cava. Arch Pharm Res 2006; 29:165–71. [15] Yang YI, Lee KT, Park HJ, Kim TJ, Choi YS, Shih Ie M, et al. Tectorigenin sensitizes paclitaxel-resistant human ovarian cancer cells through downregulation of the Akt and NFkappaB pathway. Carcinogenesis 2012;33:2488–98.

[16] Anto RJ, Maliekal TT, Karunagaran D. L-929 cells harboring ectopically expressed Re1A resist curcumin-induced apoptosis. J Biol Chem 2000;275:15601–4. [17] Arbibe L, Mira JP, Teusch N, Kline L, Guha M, Mackman N, et al. Toll-like receptor 2mediated NF-kappa B activation requires a Rac1-dependent pathway. Nat Immunol 2000;1:533–40. [18] Wang Q, Zheng XL, Yang L, Shi F, Gao LB, Zhong YJ, et al. Reactive oxygen speciesmediated apoptosis contributes to chemosensitization effect of saikosaponins on cisplatin-induced cytotoxicity in cancer cells. J Exp Clin Cancer Res 2010; 29:159. [19] Yao X, Panichpisal K, Kurtzman N, Nugent K. Cisplatin nephrotoxicity: a review. Am J Med Sci 2007;334:115–24. [20] Lee SH, Park MH, Kang SM, Ko SC, Kang MC, Cho S, et al. Dieckol isolated from Ecklonia cava protects against high-glucose induced damage to rat insulinoma cells by reducing oxidative stress and apoptosis. Biosci Biotechnol Biochem 2012; 76:1445–51. [21] Park E, Ahn G, Yun JS, Kim MJ, Bing SJ, Kim DS, et al. Dieckol rescues mice from lethal irradiation by accelerating hemopoiesis and curtailing immunosuppression. Int J Radiat Biol 2010;86:848–59. [22] Zhang C, Li Y, Qian ZJ, Lee SH, Li YX, Kim SK. Dieckol from Ecklonia cava Regulates Invasion of Human Fibrosarcoma Cells and Modulates MMP-2 and MMP-9 Expression via NF-kappaB Pathway. Evid Based Complement Alternat Med 2011;2011:140462. [23] Park SJ, Jeon YJ. Dieckol from Ecklonia cava suppresses the migration and invasion of HT1080 cells by inhibiting the focal adhesion kinase pathway downstream of Rac1ROS signaling. Mol Cells 2012;33:141–9. [24] Park SJ, Kim YT, Jeon YJ. Antioxidant dieckol downregulates the Rac1/ROS signaling pathway and inhibits Wiskott-Aldrich syndrome protein (WASP)-family verprolinhomologous protein 2 (WAVE2)-mediated invasive migration of B16 mouse melanoma cells. Mol Cells 2012;33:363–9. [25] Kwon YH, Jung SY, Kim JW, Lee SH, Lee JH, Lee BY, et al. Phloroglucinol inhibits the bioactivities of endothelial progenitor cells and suppresses tumor angiogenesis in LLC-tumor-bearing mice. PLoS One 2012;7:e33618. [26] Kong CS, Kim JA, Yoon NY, Kim SK. Induction of apoptosis by phloroglucinol derivative from Ecklonia Cava in MCF-7 human breast cancer cells. Food Chem Toxicol 2009;47:1653–8. [27] Arafa el SA, Zhu Q, Barakat BM, Wani G, Zhao Q, El-Mahdy MA, et al. Tangeretin sensitizes cisplatin-resistant human ovarian cancer cells through downregulation of phosphoinositide 3-kinase/Akt signaling pathway. Cancer Res 2009;69:8910–7. [28] Fruehauf JP, Meyskens Jr FL. Reactive oxygen species: a breath of life or death? Clin Cancer Res 2007;13:789–94. [29] Akaboshi M, Kawai K, Maki H, Akuta K, Ujeno Y, Miyahara T. The number of platinum atoms binding to DNA, RNA and protein molecules of HeLa cells treated with cisplatin at its mean lethal concentration. Jpn J Cancer Res 1992;83:522–6.

Please cite this article as: Yang Y-I, et al, Brown algae phlorotannins enhance the tumoricidal effect of cisplatin and ameliorate cisplatin nephrotoxicity, Gynecol Oncol (2014), http://dx.doi.org/10.1016/j.ygyno.2014.11.015

10

Y.-I. Yang et al. / Gynecologic Oncology xxx (2014) xxx–xxx

[30] Kang MA, So EY, Simons AL, Spitz DR, Ouchi T. DNA damage induces reactive oxygen species generation through the H2AX-Nox1/Rac1 pathway. Cell Death Dis 2012;3: e249. [31] Berndtsson M, Hagg M, Panaretakis T, Havelka AM, Shoshan MC, Linder S. Acute apoptosis by cisplatin requires induction of reactive oxygen species but is not associated with damage to nuclear DNA. Int J Cancer 2007;120:175–80. [32] Rabi T, Bishayee A. d -Limonene sensitizes docetaxel-induced cytotoxicity in human prostate cancer cells: generation of reactive oxygen species and induction of apoptosis. J Carcinog 2009;8:9. [33] Yang YI, Kim JH, Lee KT, Choi JH. Costunolide induces apoptosis in platinum-resistant human ovarian cancer cells by generating reactive oxygen species. Gynecol Oncol 2011;123:588–96. [34] Faurschou A, Gniadecki R. TNF-alpha stimulates Akt by a distinct aPKC-dependent pathway in premalignant keratinocytes. Exp Dermatol 2008;17:992–7. [35] Jung HA, Jin SE, Ahn BR, Lee CM, Choi JS. Anti-inflammatory activity of edible brown alga Eisenia bicyclis and its constituents fucosterol and phlorotannins in LPSstimulated RAW264.7 macrophages. Food Chem Toxicol 2013;59:199–206. [36] Lee SH, Kim JY, Yoo SY, Kwon SM. Cytoprotective effect of dieckol on human endothelial progenitor cells (hEPCs) from oxidative stress-induced apoptosis. Free Radic Res 2013;47:526–34. [37] Kwon TH, Kim TW, Kim CG, Park NH. Antioxidant activity of various solvent fractions from edible brown alga, Eisenia bicyclis and its active compounds. J Food Sci 2013; 78:C679–84. [38] Zhou K, Raffoul JJ. Potential anticancer properties of grape antioxidants. J Oncol 2012;2012:803294. [39] Kim AR, Shin TS, Lee MS, Park JY, Park KE, Yoon NY, et al. Isolation and identification of phlorotannins from Ecklonia stolonifera with antioxidant and anti-inflammatory properties. J Agric Food Chem 2009;57:3483–9. [40] Kuhlmann MK, Burkhardt G, Kohler H. Insights into potential cellular mechanisms of cisplatin nephrotoxicity and their clinical application. Nephrol Dial Transplant 1997; 12:2478–80. [41] Masuda H, Tanaka T, Takahama U. Cisplatin generates superoxide anion by interaction with DNA in a cell-free system. Biochem Biophys Res Commun 1994;203: 1175–80.

[42] Tsuruya K, Ninomiya T, Tokumoto M, Hirakawa M, Masutani K, Taniguchi M, et al. Direct involvement of the receptor-mediated apoptotic pathways in cisplatininduced renal tubular cell death. Kidney Int 2003;63:72–82. [43] Pedraza-Chaverri J, Maldonado PD, Medina-Campos ON, Olivares-Corichi IM, Granados-Silvestre MA, Hernandez-Pando R, et al. Garlic ameliorates gentamicin nephrotoxicity: relation to antioxidant enzymes. Free Radic Biol Med 2000;29: 602–11. [44] Gilgun-Sherki Y, Melamed E, Offen D. Oxidative stress induced-neurodegenerative diseases: the need for antioxidants that penetrate the blood brain barrier. Neuropharmacology 2001;40:959–75. [45] Wu G, Fang YZ, Yang S, Lupton JR, Turner ND. Glutathione metabolism and its implications for health. J Nutr 2004;134:489–92. [46] Zhang C, Li Y, Qian ZJ, Lee SH, Li YX, Kim SK. Dieckol from Ecklonia cava Regulates Invasion of Human Fibrosarcoma Cells and Modulates MMP-2 and MMP-9 Expression via NF-kappa B Pathway. Evid Based Complement Alternat 2011;2011. [47] Li Y, Kim SK. Utilization of seaweed derived ingredients as potential antioxidants and functional ingredients in the food industry: an overview. Food Sci Biotechnol 2011;20:1461–6. [48] Manach C, Williamson G, Morand C, Scalbert A, Remesy C. Bioavailability and bioefficacy of polyphenols in humans. I. Review of 97 bioavailability studies. Am J Clin Nutr 2005;81:230S–42S. [49] Williamson G, Manach C. Bioavailability and bioefficacy of polyphenols in humans. II. Review of 93 intervention studies. Am J Clin Nutr 2005;81: 243S–55S. [50] Carbonaro M, Grant G, Pusztai A. Evaluation of polyphenol bioavailability in rat small intestine. Eur J Nutr 2001;40:84–90. [51] Rubio L, Macia A, Motilva MJ. Impact of various factors on pharmacokinetics of bioactive polyphenols: an overview. Curr Drug Metab 2014;15:62–76. [52] Ohara M, Ohyama Y. Delivery and application of dietary polyphenols to target organs, tissues and intracellular organelles. Curr Drug Metab 2014;15:37–47.

Please cite this article as: Yang Y-I, et al, Brown algae phlorotannins enhance the tumoricidal effect of cisplatin and ameliorate cisplatin nephrotoxicity, Gynecol Oncol (2014), http://dx.doi.org/10.1016/j.ygyno.2014.11.015

Brown algae phlorotannins enhance the tumoricidal effect of cisplatin and ameliorate cisplatin nephrotoxicity.

The clinical application of cisplatin is limited due to its drug resistance and side effects. We investigated the effect of a phlorotannin-rich extrac...
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