J BIOCHEM MOLECULAR TOXICOLOGY Volume 28, Number 1, 2014

Arctigenin Enhances Chemosensitivity to Cisplatin in Human Nonsmall Lung Cancer H460 Cells through Downregulation of Survivin Expression Huan-qin Wang, Jian-jun Jin, and Jing Wang Department of Respiratory Medicine, The First Affiliated Hospital, Zhengzhou University, Zhengzhou, People’s Republic of China; E-mail: [email protected] Received 7 June 2013; revised 25 September 2013; accepted 12 October 2013

ABSTRACT: Arctigenin, a dibenzylbutyrolactone lignan, enhances cisplatin-mediated cell apoptosis in cancer cells. Here, we sought to investigate the effects of arctigenin on cisplatin-treated non-small-cell lung cancer (NSCLC) H460 cells. The 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide assay and annexin-V/propidium iodide staining were performed to analyze the proliferation and apoptosis of H460 cells. Arctigenin dose-dependently suppressed cell proliferation and potentiated cell apoptosis, coupled with increased cleavage of caspase-3 and poly(ADP-ribose) polymerase. Moreover, arctigenin sensitized H460 cells to cisplatin-induced proliferation inhibition and apoptosis. Arctigenin alone or in combination with cisplatin had a significantly lower amount of survivin. Ectopic expression of survivin decreased cell apoptosis induced by arctigenin (P < 0.05) or in combination with cisplatin (P < 0.01). Moreover, arctigenin (P < 0.05) or in combination with cisplatin (P < 0.01) induced G1/G0 cell-cycle arrest. Our data provide evidence that arctigenin has a therapeutic potential in combinaC 2013 tion with chemotherapeutic agents for NSLC.  Wiley Periodicals, Inc. J. Biochem. Mol. Toxicol. 28:39–45, 2014; View this article online at wileyonlinelibrary.com. DOI 10.1002/jbt.21533

KEYWORDS: Arctigenin; Apoptosis; Chemoresistance; Cisplatin; Non-small-cell lung cancer (NSCLC).

INTRODUCTION Cisplatin, an anticancer drug containing platinum, is widely used for the treatment of solid tumors, including non-small-cell lung cancer (NSCLC) [1]. Cis-

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Correspondence to: Dr. Jing Wang. 2013 Wiley Periodicals, Inc.

platin, in combination with one third-generation anticaner drug, such as vinorelbine, gemcitabine, or taxanes, represents the standard regimen used in the firstline treatment of advanced NSCLC [2]. Evaluated in multiple phase III trials, cisplatin showed consistent superior efficiency compared with other regimens [2]. However, the development of resistance to cisplatin is common during the treatment of NSCLC [2]. Decreased apoptotic activity was identified as the most important mechanism for cisplatin resistance in NSCLC [3]. Apoptosis defects in response to chemotherapy in cancer cells are due to the overexpression of antiapoptotic proteins and/or loss of proapoptotic proteins [4–6]. Thus, modulation of these apoptosis-associated proteins could help to overcome chemoresistance during cancer treatment. Survivin is a member of the inhibitor of apoptosis protein family that is involved in the regulation of cell survival and proliferation [7]. Survivin is selectively expressed in most human cancers, including NSCLC, and is associated with a poor prognosis in patients with NSCLC [8]. Moreover, survivin is suggested to confer tumor cell resistance to anticancer agents and ionizing radiation [9]. Survivin, therefore, has become an attractive target for novel anticancer interventional agents [10]. In recent years, great research effort has been directed toward counteracting survivin [11, 12]. Several preclinical studies have shown that inhibition of survivin expression or function could attenuate tumor growth, increase spontaneous and induced apoptosis, and sensitize tumor cells to anticancer agents [13]. Arctigenin, a dibenzylbutyrolactone lignan, is abundantly produced by Bardanae fructus, Arctium lappa L, Saussurea medusa, Torreya nucifera, and Ipomea cairica [14]. Arctigenin possesses anti-inflammation and antitumor activities [14–16]. Recently, it has been demonstrated that arctigenin dramatically enhances cisplatin-mediated cell apoptosis in cancer cells 39

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through suppressing the activation of signal transducer and activator of transcription 3 (STAT3; 16). These results indicate that arctigenin has a therapeutic potential in combination with chemotherapeutic agents for cancer treatment. In the present study, we sought to investigate the effects of arctigenin on cisplatin-mediated cytotoxicity in NSCLC cells.

MATERIALS AND METHODS

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Cell Proliferation Assay After the indicated treatment as described above, cell proliferation was analyzed with a 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Promega, Madison, WI) according to the manufacturer’s protocol. The absorbance was measured at 570 nm using a microplate reader. All assays were performed at least in triplicates. For calculation of the relative proliferation rate, the mean absorbance in the DMSO-treated control cells was set as 100%.

Cell Culture Human lung large cell carcinoma H460 cells were purchased from the American Type Culture Collection (Manassas, VA). Cells were cultured in RPMI 1640 medium (Life Technologies, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37◦ C in a 5% CO2 incubator.

Drug Treatment Arctigenin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and dissolved in dimethyl sulfoxide (DMSO). For cell proliferation assay, H460 cells were exposed to different concentrations of arctigenin (1–20 μM) for 72 h. For apoptosis analysis and cell-cycle analysis, cells were treated with arctigenin at a concentration of 10 μM for 48 h.

Plasmid Construction and Transfection TRIzol reagent (Invitrogen, Carlsbad, CA) was employed to isolate total RNA from H460 cells according to the manufacture’s protocol. Human survivin (GenBank NM 001168) full-length cDNA (429 bp) was synthesized by reverse transcription of the extracted total RNA and amplified by polymerase chain reaction (PCR). The PCR primers for survivin were as follows: forward, 5 -GCCGAAGCTTACCATGGGTGCC-3 ; and reverse, 5 -GCGCCTCGAGTCAATCCATGGC-3 . After digestion with Hind III and Xho I, the resulting restriction fragments were cloned into the Hind III/Xho I sites of the pcDNA3.1 (+) expression vector (Invitrogen). The constructed plasmids (pcDNA3.1-survivin) were verified by DNA sequencing. For overexpression of survivin, H460 cells were transfected with vector or pcDNA3.1-survivin by Lipofectamine 2000 (Invitrogen) and incubated for 24 h. The cells were treated with drugs for another 48 h and collected for apoptosis assay.

Apoptosis Analysis by Flow Cytometry After the indicated treatment, cells were harvested by trypsinization, washed three times in phosphate buffered saline (PBS), and resuspended in 0.5 mL PBS. Immediately after resuspension of the cells, propidium iodide (PI) and a fluorescein isothiocyanate (FITC)conjugated monoclonal antibody specific for Annexin V (KaiGi Technology, Guangzhou, People’s Republic of China) were incubated with the cells at 4◦ C for a period of 30 min. Cell apoptosis was measured using flow cytometry (Becton Dickinson Biosciences, San Jose, CA).

Western Blot Analysis Primary antibodies employed were: anti-cleaved caspase-3 (Cell Signaling Technology, Beverly, MA), anti-cleaved poly ADP ribose polymerase (PARP) (Cell Signaling Technology), anti-survivin (Santa Cruz Biotech), and anti-β-actin (Santa Cruz Biotech). Cells with indicated treatment were lysed in a 10-mM Tris buffer (pH 7.4) containing 1% sodium dodecyl sulfate (SDS) and complete protease inhibitors (Roche, Mannheim, Germany). 20 μg of the lysates were separated by SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA), and probed with the primary antibodies. After incubation with appropriate horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotech), blots were visualized with an enhanced chemiluminescent detection system (Amersham Biosciences, Piscataway, NJ). The intensity of each band was measured by densitometric analysis using the Quantity One software (Bio-Rad, Hercules, CA). For calculation of the relative expression level, the mean ratios of target protein/β-actin were normalized to those in DMSO-treated controls cells (100%).

Cell-Cycle Analysis Cells were trypsinized, washed with PBS, fixed in 70% ethanol, treated with 10 μl RNase (10 μg/mL), J Biochem Molecular Toxicology

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FIGURE 1. The effects of arctigenin on H460 cell proliferation. (A) H460 cells were treated with different concentrations of arctigenin (1–20 μM) for 72 h. MTT assay was used to analyze the effects of arctigenin on the proliferation of H460 cells. (B) Arctigenin increased cisplatin-induced inhibition of H460 cell proliferation. H460 cells were exposed to indicated drugs for 72 h. MTT assay was performed to analyze the proliferation of H460 cells. Data are presented as mean ± SD (n = 3).*P < 0.05, **P < 0.01 versus control cells.

and finally stained with 10 μL PI (1 mg/mL). Small cellular debris was gated out of the data analysis. The cellular DNA content of treated cells was analyzed by flow cytometry (Becton Dickinson Biosciences).

Statistical Analysis Data are presented as means ± standard deviation (SD). All statistical calculations were carried out using SPSS.11 software (SPSS, Chicago, IL). The difference among the means of multiple groups was analyzed by Student’s t-test or one-way analysis of variance (ANOVA) followed by the Tukey test. A difference was defined as significant at P < 0.05 in Student’s t-test or one-way ANOVA.

FIGURE 2. The effects of arctigenin on H460 apoptosis. H460 cells were treated with arctigenin at a concentration of 10 μM for 48 h. (A) Annexin V/PI double staining were performed to measure the apoptosis ratio in arctigenin-exposed cells. Representative flow cytometry profiles and percentages of apoptotic cells (annexin-Vpositive and PI-negative cells in lower right quadrant) are shown. (B) Western blot analysis was used to analyze the expression of cleaved caspase-3 and PARP in arctigenin-treated H460 cells. Representative blots are shown from three independent experiments with almost identical results. Quantitative densitometric analysis of the relative expression of target proteins is shown in the lower panel. The mean ratio of target protein/β-actin in control cells was set as 1. Data are presented as mean ± SD (n = 3). *P < 0.01 versus control cells.

cisplatin-mediated inhibition of H460 proliferation (P < 0.01 vs. cisplatin alone-treated cells; Figure 1B).

RESULTS Arctigenin has Antiproliferative Effects on H460 Cells 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay revealed that arctigenin inhibited the proliferation of H460 cells in a dose-dependent manner (Figure 1A). Arctigenin at a concentration of 10 μM resulted in about 50% reduction of proliferation, compared with untreated control cells. The proliferation of H460 was decreased by 28% and 65% in 2 and 10 μM of cisplatin, respectively. Notably, 10 μM of arctigenin profoundly elevated J Biochem Molecular Toxicology

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Arctigenin Induces Apoptosis in H460 Cells We then examined whether arctigenin-mediated proliferation inhibition was due to the induction of apoptosis. After treatment with 10 μM arctigenin for 48 h, the percentage of annexin-V-FITC-positive cells was increased to 9.5 ± 2.0%, compared with 2.7 ± 1.2% in control (Figure 2A). Moreover, Western blot analysis confirmed such induction of apoptosis, and the levels of cleaved caspase-3 and PARP were markedly elevated upon the arctigenin treatment (Figure 2B).

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FIGURE 3. Arctigenin sensitizes H460 cells to cisplatin-induced apoptosis. H460 cells were exposed to indicated drugs for 48 h. Annexin-V/PI double staining was used to measure the apoptosis ratio. Representative flow cytometry profiles and quantitative analysis are shown in (A) and (B). Data are presented as mean ± SD (n = 3). *P < 0.05, **P < 0.01 versus control cells.

Arctigenin Sensitizes H460 Cells to Cisplatin-Induced Apoptosis Apoptosis analysis revealed that after a 48-h treatment, cisplatin alone at 2 and 10 μM caused 8.4 ± 1.9% and 15.6 ± 2.3% of H460 cells to undergo apoptosis, respectively (Figure 3). Interestingly, the combination with arctigenin significantly enhanced the cytotoxicity of relatively low-dose cisplatin. There was 27.2 ± 3.1% apoptosis seen in H460 cells treated with a combination of 2 μM cisplatin and 10 μM arctigenin (Figure 3).

Downregulation of Survivin Contributes to Arctigenin-Induced Chemosensitization It has been documented that survivin plays a critical role in apoptosis resistance in NSCLC cells [17]. Therefore, we checked whether the chemosensitive effect of arctigenin was mediated through the downregualtion of survivin. As shown in Figure 4A, treatment with arctigenin alone for 48 h significantly reduced the protein expression of survivin in H460 cells. Although 2-μM cisplatin only marginally suppressed the survivin expression, its combination with arctigenin led to more than 50% reduction in the survivin protein level (Figure 4A). To further investigate the effects of survivin on arctigenin-induced apoptosis in H460 cells, pcDNA3.1-survivin, or pcDNA3.1 empty vector was transfected into H460 cells. The protein level of survivin in H460 cells transfected with pcDNA3.1survivin was about 10-fold of that in empty vectortransfected cells (Figure 4B). Most importantly, flow

cytometry analysis revealed that the overexpression of survivin significantly blocked the induction of apoptosis by arctigenin alone (P < 0.05) or in combination with cisplatin (P < 0.01), compared with mock-transfected cells (Figure 4C).

Effects of Arctigenin on Cell-Cycle Distribution in Cisplatin-Treated H460 Cells To determine the effects of arctigenin on cell-cycle progression of NSCLC cells, H460 cells were cultured in the presence of arctigenin alone or in the combination with cisplatin for 48 h. The cell-cycle distribution was determined by flow cytometry. Cisplatin at the dose of 2 μM did not show statistical significant effects on the cell-cycle distribution (Figure 5). Incubation with 10 μM of arctigenin or 10 μM of arctigenin together with 2 μM of cisplatin resulted in a G0/G1 cell-cycle block, as determined by an increase of the G0/G1 population and by a corresponding reduction of the S and G2/M populations (Figure 5).

DISCUSSION It has been reported that arctigenin shows cytotoxicity against several human cancer cells such as A549 NSCLC, HepG2 liver cancer, and KATO III stomach cancer cells, while exerting little or no cytotoxic effect in normal cells [18]. This high specificity for cancer cells makes arctigenin as a promising anticancer agent. Here, we showed that arctigenin J Biochem Molecular Toxicology

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FIGURE 4. Inhibition of survivin confers arctigenin-mediated chemosensitivity of H460 cells to cisplatin. H460 cells were exposed to indicated drugs for 48 h. (A) Western blot analysis was used to examine the expression of survivin in H460 cells with indicated treatment. Quantitative densitometric analysis of the relative expression of survivin is shown in the lower panel. The mean ratio of survivin/β-actin in control cells was set as 100%. (B) pcDNA3.1-survivin or pcDNA3.1 empty vector was transfected into H460 cells for 72 h. The expression levels of survivin in H460 cells-transfected with pcDNA3.1-survivin or pcDNA3.1 empty vector were examined by Western blot analysis (upper panel). The mean ratio of survivin/β-actin in control cells (H460 cells without drug treatment was used as control cells) was set as 1. Quantitative densitometric analysis of the relative expression of survivin is shown in the lower panel. (C) Annexin V/PI double staining was performed to analyze the apoptosis ratio in H460 cells with indicated drugs. Data are presented as mean ± SD (n = 3). *P < 0.05; **P < 0.01 versus indicated control.

treatment inhibited cell proliferation and induced apoptosis in H460 cells. Recently, arctigenin has been found to promote cisplatin-induced cell death in cancer cells [16]. Consistently, we revealed that arctigenin profoundly enhanced cisplatin-mediated H460 cell apoptosis. These findings highlight the chemosensitive activity of arctigenin.

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Survivin, a prominent anti-apoptotic molecule, is expressed widely in different types of cancers [19]. Overexpression of survivin leads to uncontrolled cancer cell growth and drug resistance [20]. Efficient inhibition of survivin expression and its functions renders tumor cells more sensitive to anticancer drugs, leading to cell apoptosis [21]. Recent studies showed that survivin is overexpressed [22, 23] and is associated with chemoresistance in NSCLC [24]. We explored the role of arctigenin on survivin expression in H460 cells. Arctigenin alone or in combination with cisplatin significantly suppressed the survivin expression, but not cisplatin alone. Consistently, overexpression of survivin remarkably attenuated arctigenin-induced apoptosis in H460 cells with or without cisplatin treatment, indicating that the downregulation of survivin confers arctigenin-mediated tumor suppressive effects and chemosensitization activity. It has been suggested that arctigenin inhibits survive expression via ant/betacatarhine signaling pathway [25]. Further investigation is needed for addressing the molecular mechanism of arctigenin-mediated suppression of survivin in NSCLC. The inhibition of survivin expression by arctigenin is also observed in SW480 human colon cancer cells [25], indicating that the inhibition of survivin might be a strategy for arctigenin-mediated antitumor activity. Arctigenin induces G0/G1 cell-cycle arrest by blocking the phosphorylation of Rb through regulating cell cycle regulatory proteins in human gastric cancer cells [26]. We analyzed the effects of arctigenin on the cell-cycle progression of H460 cells with or without cisplatin treatment. Arctigenin alone significantly enhanced the accumulation of H460 cells in the G0/G1 phase. Moreover, the induction of cell-cycle arrest was potentiated by the combination of arctigenin and cisplatin. These results suggest that arctigenin-mediated induction of cell-cycle arrest may partially account for its chemosensitization in NSCLC cells. However, arctigenin is also shown to induce G2/M arrest in SW480 human colon cancer cells as well as AGS human gastric cancer cells [27]. It seems that arctigenin-mediated cell-cycle arrest is cell-context dependent. A major limitation of this study is that the data are obtained from only one NSCLC cell line. Therefore, it is still unclear whether arctigenin exerts similar chemosensitive effects on other NSCLC cell lines. The anticancer activity and side effect of arctigenin in animal models also need further investigation. Taken together, our results demonstrate that arctigenin induces G0/G1 cell-cycle arrest and apoptosis and enhances cisplatin-mediated cytotoxicity in H460 cells. Therefore, arctigenin may have therapeutic implications against NSCLC.

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FIGURE 5. The effects of arctigenin on cell-cycle distribution of H460 cells. The analysis of changes in cell cycle was quantified by flow cytometry after PI staining in H460 cells treated with 10 μM of arctigenin in the presence or absence of 2 μM of cisplatin. Dead cells were gated out, leaving only live cells to be analyzed for cell cycle. Histograms (A) show a representative analysis of cell-cycle distribution, and bar graphs (B) represent the quantitative analysis from three independent experiments. Data are presented as mean ± SD (n = 3). *P < 0.05; **P < 0.01 versus control cells.

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Arctigenin enhances chemosensitivity to cisplatin in human nonsmall lung cancer H460 cells through downregulation of survivin expression.

Arctigenin, a dibenzylbutyrolactone lignan, enhances cisplatin-mediated cell apoptosis in cancer cells. Here, we sought to investigate the effects of ...
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