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Xanthohumol inhibits cell cycle progression and proliferation of larynx cancer cells in vitro
Chemico-Biological Interactions 240 (2015) 110e118
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Xanthohumol inhibits cell cycle progression and proliferation of larynx cancer cells in vitro ska-Brych a, *, Sylwia Katarzyna Kro l b, Adrianna Sławin ska c, Andrzej Stepulak b, ska-Graniczka b, Barbara Zdzisin Magdalena Dmoszyn a Mariusz Gagos a b c
Department of Cell Biology, Maria Curie-Sklodowska University, Akademicka 19, 20-033 Lublin, Poland Department of Biochemistry and Molecular Biology, Medical University of Lublin, Chodzki 1, 20-093 Lublin, Poland Department of Virology and Immunology, Maria Curie-Sklodowska University, Akademicka 19, 20-033 Lublin, Poland
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 April 2015 Received in revised form 19 July 2015 Accepted 11 August 2015 Available online 20 August 2015
Xanthohumol (XN), a prenylflavonoid derived from the hop plant (Humulus lupulus L.) has been found to exhibit a broad spectrum of biological properties, including anti-cancer activity. In this study, the mechanisms involved in anti-cancer activity of XN in human RK33 and RK45 larynx cancer cell lines were investigated. The effect of XN on the viability of larynx cancer and normal cells (human skin fibroblasts HSF and rat oligodendroglia-derived cells, OLN-93) was compared. Additionally, the influence of XN on proliferation, cell cycle progression, induction of apoptosis in larynx cancer cells, as well as the molecular mechanisms underlying in these processes were analyzed. XN promoted the reduction of cell viability in cancer cells, but showed low cytotoxicity to normal cells. The decrease in cell viability in the cancer cells was coupled with induction of apoptosis via two pathways. The mechanisms involved in these effects of XN were associated with cell growth inhibition by induction of cell cycle arrest in the G1 phase, increased p53 and p21/WAF1 expression levels, downregulation of cyclin D1 and Bcl-2, and activation of caspases9, -8, and -3. Moreover, this compound inhibited phosphorylation of ERK1/2, suggesting a key role of the ERKs pathway in the XN-mediated growth suppressing effects against the studied cells. These results indicate that XN could be used as a potential agent for the treatment of patients with larynx cancer. © 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Xanthohumol (XN) Larynx cancer cells Apoptosis Cell cycle arrest Extracellular signal regulated kinase 1 and 2 (ERK1/2)
1. Introduction Laryngeal squamous cell carcinoma (LSCC) is the most common malignancy of the head and neck worldwide [28]. According to the American Cancer Society, LSCC accounts for 10% of cancers in men and 4% in women [2]. Despite recent advance and achievements in diagnostic strategies and oncological treatment, LSCC is still an important therapeutic problem. The 5-year survival rates for laryngeal cancer patients with regional and metastatic LSCC demonstrated a significant decrease within last three decades, irrespective of the applied therapy [9,25]. Therefore, there is a need for discovery of novel, more effective therapeutic agents, alternative to traditionally used cisplatin (cis-diammine-dichloridoplatinum, CDDP) and 5-fluorouracil (5-FU), to improve the
* Corresponding author. skaE-mail address: [email protected] (A. Sławin Brych). http://dx.doi.org/10.1016/j.cbi.2015.08.008 0009-2797/© 2015 Elsevier Ireland Ltd. All rights reserved.
currently available treatment strategies and survival rates, particularly for patients with advanced cases of LSCC [30]. One of the most interesting and encouraging approaches is the use of natural, bioactive substances from plants as potential chemopreventive and chemotherapeutic agents in the therapy of several human cancers. There is still increasing evidence that some plant secondary metabolites could be considered as promising anticancer drugs [33,34,37,38]. Xanthohumol (XN, (E)-1-[2,4-Dihydroxy-6-methoxy-3-(3methylbut-2-enyl)phenyl]-3-(4-hydroxyphenyl)prop-2-en-1-one) is a prenylated chalcone, a representative of flavonoids, a large group of plant metabolites. XN is naturally distributed in Humulus lupulus L. (Cannabaceae) as a main component of the female inflorescences e up to 1% in dry hop cones [54]. Recently, a growing number of reports have demonstrated that XN elicits a wide spectrum of anti-cancer and chemopreventive activity such as inhibition of the metabolic activation of pro-carcinogens, induction of carcinogen-detoxifying enzymes, and inhibition of tumor growth
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in vitro [46]. XN has been shown to inhibit growth and proliferation of human breast (MCF-7) [49,54], colon (HT-29), ovarian (A2780), and prostate (DU145, PC-3) cancer cells [54,49,7] as well as Bchronic lymphocytic leukemia [32], hepatocellular carcinoma [15], and medullary thyroid cancer cells [8]. Moreover, pro-apoptotic activity of XN has also been observed in human breast cancer [47], prostate cancer [7], B-chronic lymphocytic leukemia [32], and malignant glioblastoma cells [18]. Besides these effects, it has been reported as a strong inhibitor of migration, invasion, and angiogenesis in hematological malignancies [11,36]. More importantly, this compound has additionally been shown to modulate radio- and chemosensitivity of cancer cells [4,27]. Taking into account its multiple antitumor functions and relatively nontoxic nature, XN could be a very promising drug for further in vivo investigation and potential application in oncology. The aim of the study was to assess the anti-cancer potential of XN in LSCC in vitro and mechanisms underlying its activity. To the best of our knowledge, here we report for the first time the anticancer effect of XN in LSCC.
(OLN-93) or 5 104 cells/ml (HSF) and left overnight to attach. Then, the culture medium was removed and the cells were treated with serial dilutions of XN (0.1e100 mM) in a fresh culture medium with 10% FBS or cultured in a medium alone as a control. Cell viability was determined after 48 h by the MTT assay, in which the yellow tetrazolium salt (MTT 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide) is reduced by mitochondrial dehydrogenase of viable cells to purple, insoluble crystals of formazan. The cells were incubated for 3 h with the MTT solution (5 mg/ml in PBS with Ca2þ/Mg2þ) (SigmaeAldrich) at 37 C. Then, formazan crystals were solubilized in lysing buffer (10% SDS in 0.01 N HCl) overnight at room temperature (RT). The product was quantified by measurement of absorbance at a 570 nm wavelength with the use €nnedorf, of an Infinite M200 Pro microplate reader (Tecan, Ma Switzerland) and the data were analyzed by Magellan™ Software Infinite M200 (Tecan, Switzerland). All experiments were performed in triplicate and yielded similar results.
2. Materials and methods
RK33 and RK45 cells were seeded on 96-well microplates at a density of 1000 cells/well (1 104/ml) and 3000 cells/well (3 104/ ml), respectively, and left overnight to attach. Then, the culture medium was removed and the cells were treated with serial dilutions of XN (1, 5, 10, 25, or 50 mM) in a fresh culture medium with 10% FBS. Control cells were cultured in a medium without XN. Cell proliferation was assessed after 48 h with the use of a Cell Proliferation Elisa BrdU kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's instructions. All experiments were performed in triplicate and yielded similar results.
2.1. Cell line and chemicals
2.3. Cell proliferation assessment
Human larynx cancer cells RK33 and RK45 were derived from a larynx squamous cell carcinoma taken from patients after total laryngectomy and established as stable cell lines as previously described [42]. The RK33 and RK45 cell lines were cultured in RPMI 1640 (SigmaeAldrich, St. Louis, USA) supplemented with 10% heatinactivated fetal bovine serum (FBS) (SigmaeAldrich), penicillin (100 U/ml) (SigmaeAldrich), and streptomycin (100 mg/ml) (SigmaeAldrich). Cultures were maintained at 37 C in a humidified atmosphere of 95% air and 5% CO2. The medium was replaced at 2e3-day intervals. Sub-confluent cells were usually rinsed with a phosphate-buffered solution (PBS) without Ca2þ/Mg2þ (SigmaeAldrich) and harvested with 0.25% Trypsin-EDTA (SigmaeAldrich). Human skin fibroblasts (HSF) were a laboratory strain established by an outgrowth technique from skin explants of a young person (healthy volunteer, employee of the Department of Virology and Immunology; the skin explants were obtained with the person' written informed consent). HSF were cultured in Dulbecco's modified Eagle Medium (SigmaeAldrich) containing 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin. Rat oligodendroglia-derived cells (OLN-93) were obtained from the Institute of Agricultural Medicine (Lublin, Poland) and were grown in a 1:1 mixture of Dulbecco's modified Eagle Medium and Ham's F12 Medium (SigmaeAldrich) supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin, 100 mg/ml streptomycin. The cells were maintained in a humidified incubator with 5% CO2 in air at 37 C. Xanthohumol (SigmaeAldrich) was dissolved in dimethyl sulfoxide (DMSO) and stored at 20 C until use. The final concentration of DMSO in all experiments was less than 0.01%, and all treatment conditions were compared with controls. U0126, a pharmacological inhibitor of MEK1/2 and PMA (Phorbol 12myristate 13-acetate), an activator of protein kinase C were also purchased from SigmaeAldrich.
RK33 and RK45 cells were seeded on 6-well plates (Nunc) at a density of 3 105 cells/ml and left overnight to attach. Then, the culture medium was removed and the cells were exposed to the selected dilutions of XN (0.1e10 mM) in a fresh culture medium with 10% FBS or cultured in a medium alone as a control. After 48 h, the treated cells were stained with propidium iodide (PI) using the PI/ RNase Staining Buffer (BD Biosciences, BD Pharmingen™, USA), according to the manufacturer's instructions. Briefly, the cells were harvested and centrifuged at 366 g for 5 min at RT, and the pellets were fixed in ice-cold 80% ethanol overnight at 20 C. Following fixation, the cells were centrifuged at 366 g for 5 min at 4 C, washed twice in PBS, and then incubated with 0.5 ml PI/RNase solution per 1 106 cells for 15 min in darkness at RT. Next, the stained cells were analyzed by the flow cytometry technique using a flow cytometer FACSCalibur™ (BD Biosciences, USA) equipped with a 488-nm argon-ion laser to assess the percentage of cells in phases G0/G1, S, G2/M, based on the amount of PI incorporated into DNA strains, and generate histograms of DNA distribution. The PI fluorescence intensity of individual nuclei was determined and at least 10 000 events were measured within an acquisition rate >60 events/second. Cell cycle analyses were performed with the use of non-commercial software Cylchred Version 1.0.2 for Windows (source: University of Wales, UK) and WinMDI 2.9 for Windows (source: facs.scripps.edu/software.html). All experiments were performed in triplicate and yielded similar results.
2.2. Cell viability (MTT assay)
2.5. Hoechst 33258 and SYTOX Green staining
The cancer cells and normal cells were seeded on 96-well microplates (Nunc, Langenselbold, Germany) at a density of 1 104 cells/ml (RK33), 3 104 cells/ml (RK45) and 1 104 cells/ml
RK33 cells (3 105 cells/ml) were seeded in 6-well plates and incubated with XN (1, 5, 10, or 15 mM) for 48 h. After that, the cells were detached with an Accutase solution (SigmaeAldrich) and
2.4. Cell cycle progression analysis
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harvested by centrifugation at 366 g for 5 min. The cell pellets were then washed with PBS and incubated for 15 min in a solution of Hoechst 33258 (5 mg/ml, Invitrogen, USA) and SYTOX Green (0.1 mM, Invitrogen, USA). After the incubation time, the cell suspension was placed on a microscope slide and covered with a coverslip. At least 500 cells in randomly selected microscopic fields were scored under a fluorescence microscope (Leica, Wetzlar, Germany) and categorized as normal, apoptotic, or necrotic. Live cells had a structurally normal blue nucleus. Apoptotic cells (with condensed, fragmented, or abnormally shaped nuclei) exhibited bright blue fluorescence. Cells exhibiting green fluorescent nuclei were interpreted as necrotic. 2.6. Caspase-3 activity assessment RK33 cells were seeded on 6-well plates at a density of 3 105 cells/ml and left overnight to attach. Then, the culture medium was removed and the cells were treated with serial dilutions of XN (1e15 mM) in a fresh culture medium with 10% FBS. After 48 h of the treatment, the cells were determined utilizing a PE Active Caspase-3 Apoptosis Kit (BD Biosciences, BD Pharmingen™, USA) according to the manufacturer's instructions. Briefly, the cells were harvested and washed twice with PBS, fixed and permeabilized using the Cytofix/Cytoperm Solution, then washed twice in the Perm/Wash Buffer prior to staining with rabbit monoclonal PE-conjugated anti-active caspase-3 antibody. Labeled cells were analyzed by the flow cytometry technique using a flow cytometer FACSCalibur™ equipped with a 488-nm argon-ion laser operating with CellQuest software to quantitatively assess the caspase-3 activity. All experiments were performed in triplicate and yielded similar results.
were incubated with stripping buffer (100 mM b-mercaptoethanol, 2% SDS, 62.5 mM TriseHCl, pH 6.8) for 20 min at 50 C, then washed, blocked, and incubated overnight at 4 C with primary antibodies: anti-ERK 2 or anti-b-Actin (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA). 2.8. Statistical analysis The data were presented as the mean value and standard error. Statistical analysis was performed with the one-way ANOVA with Tukey's post hoc test (GraphPad Prism5, GraphPad Software, Inc, San Diego, California, USA). Significance was accepted at P < 0.01. 3. Results 3.1. Effect of XN on the cell viability The survival rates of RK33 and RK45 cells treated with XN was assessed by an MTT assay after 48 h. As shown in Fig. 1A, the exposure to the increasing concentrations of XN (0.1e100 mM) caused a dose-dependent reduction in cell viability of both larynx cancer cell lines. However, XN was more effective for RK33 than RK45 cells. In RK33 culture, a statistically significant effect was seen at the 1 mM concentration (P < 0.01) and in RK45 cells at the 5 mM concentration (P < 0.01). Incubation with 15 mM and 25 mM induced
2.7. Protein extraction and Western blot analysis Cancer cells were seeded on 6-well plates at a density of 3 105 cells/ml and left overnight to attach. Then, the growth medium was changed and the cells were cultured with or without increasing concentrations of XN at different lengths of time (4 h for ERK1/2 activation and 48 h for p21, p53, cyclin D1, Bcl-2, and caspase-8 and caspase-9 analysis). The cells were harvested and lysed in RIPA buffer (1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 20 mM NaF, 0.5 mM DTT, 1 mM PMSF, and protease inhibitor cocktail in PBS pH 7.4) and then centrifuged at 8000 g for 10 min at 4 C. The total cellular protein concentration was quantified using the Bradford protein assay reagent (BioRad). For Western-blot analysis, supernatants of RIPA cell lysates were solubilized in 5 Laemmli Sample Buffer (50% glycerol, 10% SDS, 300 mM TriseHCl pH 6.8, 0.05% bromophenol blue, 6.25% b-mercaptoethanol) and boiled for 5 min at 100 C. Equal amounts of protein extracts were electrophoresed on 10% SDSPAGE and transferred onto a PVDF membrane (Millipore, USA). After the transfer, the membranes were blocked for 1 h at RT with 5% non-fat dry milk/TBS/0.1% Tween (SigmaeAldrich) and incubated overnight at 4 C with the following primary antibodies: antip21Waf1/Cip1, anti-p53, anti-cyclin D1, anti-Bcl-2 (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA), anti-Phospho-p44/p42 MAPK (ERK1/2) (Thr202/Tyr204) (1:2000, Cell Signaling Inc., USA), anticaspase-8, and anti-caspase-9 (1: 4000, Santa Cruz Biotechnology, Santa Cruz, CA). After incubation with the primary antibodies and washing with TBS/0.1% Tween, appropriate horseradish peroxidase-conjugated rabbit or mouse secondary antibodies (1:2000, Santa Cruz Biotechnology, Santa Cruz, CA) were added. Detection of protein bands was performed using an enhanced chemiluminescence (ECL) LUMI-LightPLUS Western Blotting Substrate Kit (Roche Diagnostics, Germany). For stripping, membranes
Fig. 1. Cytotoxic effect of XN on larynx cancer cells (A) and normal cells (B) after 48 h treatment with various concentrations of XN (0.1e100 mM). The cell viability was measured by the MTT assay. The results represent the mean ± SD of three independent experiments. *Statistically significant at P < 0.01 in comparison to the control (untreated cells).
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a decrease in the number of viable cells by 54%e71% and 46%e56% in RK33 and RK45, respectively. The highest inhibitory activity of XN was achieved at 100 mM. To study whether the XN-dependent suppression was selective for tumor cells, the impact of XN on the viability of non-malignant human skin fibroblast (HSF) and rat oligodendrocytes (OLN93) was also examined. As shown in Fig. 1B, the normal cells were less sensitive to the action of XN in comparison to the cancer cells. At the concentrations from 0.1 to 10 mM, this compound was almost non-toxic. Only high doses of XN (25e100 mM) exerted a pronounced inhibitory effect. These results indicate that XN may selectively target larynx cancer cells but spare normal cells, which is a highly attractive property of potential antitumor drugs. 3.2. XN suppresses cell proliferation and induces alteration in cell cycle distribution of larynx cancer cells RK33 and RK45 cancer cells were cultured in a complete medium in the absence (control) or presence of selected concentrations of XN for 48 h. The influence of XN on the cell proliferation was assessed by the BrdU assay. As shown in Fig. 2, XN diminished BrdU incorporation into cells in a dose-dependent manner. The XN concentrations, at which a significant inhibition in DNA synthesis was initially observed, were 1 mM for RK33 (P < 0.01) and 10 mM for RK45 cells (P < 0.01), indicating lower antiproliferative activity of XN against the RK45 cell line. The maximal dose of XN applied (50 mM) resulted in a 77% and 44% decrease in the number of proliferative cells in the RK33 and RK45 culture, respectively. Because cell cycle perturbation plays a critical role in tumorigenesis, and mitotic division, differentiation, and apoptosis are all cell cycle-dependent, we additionally analyzed the effect of XN on the cell cycle progression of both cell lines. Incubation with XN (0.1e10 mM) for 48 h led to a dose-dependent accumulation of cells in the G1 phase of the cell cycle. An increase in the G1 cell subpopulation was accompanied by cell reduction in the G2 phase. After treatment with 10 mM of XN, the percentage of cells in the G1 phase increased from 59.8% and 47.4% in the controls to 79% and 56.3% in RK33 and RK45, respectively (Fig. 3A, B). Our data suggest that the XN growth inhibitory effect could be related to inhibition of cell cycle progression.
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To further elucidate the mechanisms of the anticancer potential of XN, the more susceptible RK33 cells were used in the subsequent studies. 3.3. XN induces extrinsic and intrinsic apoptotic pathways in larynx cancer cells For determining whether the loss of cell viability observed in RK33 cultures with the presence of XN could be due to the apoptotic and/or necrotic mechanism, the cells were analyzed using double staining with Hoechst 33258 and SYTOX Green dyes. Fluorescence microscopy revealed an unchanged morphology of the nuclei in the untreated cells as well as chromatin fragmentation and appearance of apoptotic bodies in the XN-treated cells, indicating pro-apoptotic activity of this agent (Fig. 4AeF). After 48 h, at the concentrations of 1 mM, 5 mM, or 10 mM, the rate of apoptosis was higher than that of necrosis (20.7% and 10.31%, respectively at 10 mM). However, when the cells were incubated with 15 mM of XN, the drug was a stronger inducer of necrosis. At this concentration, approximately 26% of the cell population underwent necrosis, whereas 22% of the cell fraction died by apoptotic cell death (Fig. 4F). To establish whether the XN-mediated apoptosis was a caspasedependent process, we performed additional studies. The results obtained from flow cytometry clearly demonstrated that XN after 48 h caused a dose-dependent increase in the cell population positive for the presence of an active form of executor caspase-3 (Fig. 5A), reaching a maximum at 10 mM. At this concentration, the activity of caspase-3 was 6,75-fold higher than in the control cells. Moreover, XN-induced caspase-3 activity was dependent on the activation of two upstream initiator caspases, caspase-8 and caspase-9, as evidenced by a significant decrease in the procaspase8 and procaspase-9 levels and elevated expression of their active forms (Fig. 5B). Furthermore, the effect of XN on caspase-9 cleavage was correlated with reduced expression of an anti-apoptotic protein Bcl-2 (a key regulator of mitochondrial permeability and cyt c release into the cytosol); (Fig. 5C, D). Our data therefore suggest that XN may trigger apoptosis of RK33 cells by initiation of an intrinsic (caspase-9-dependent) pathway, and at least in part by extrinsic (caspase-8-dependent) apoptotic signaling. 3.4. Effect of XN on the levels of the cell cycle regulators To further study the molecular events leading to cell death and growth inhibition following XN treatment, we evaluated the expression levels of p53, p21, and cyclin D1 proteins (key molecular markers associated with the G1/S phase) in RK 33 cells using Western blot analysis. As shown in Fig. 6, after 48 h, XN (1e15 mM) affected the expression of all studied proteins in the RK33 cells. The dose of 1 mM slightly increased the expression of p53 and p21, compared to the control. Marked induction of these proteins was detected after incubation with 5 mM and 10 mM XN. In addition, XN decreased the cyclin D1 protein level and this decrease was dosedependent. A noticeable reduction of cyclin D1 expression was even seen at 1 mM. 3.5. XN inhibits ERK1/2 phosphorylation
Fig. 2. Effect of XN on cell proliferation. RK33 (A) and RK45 (B) cells were treated with increasing concentrations of XN for 48 h. Cell proliferation was determined by the BrdU incorporation assay. The results represent the mean ± SD of three independent experiments. *Statistically significant at P < 0.01 in comparison to the control (untreated cells).
Up-regulated signaling through the extracellular signalregulated kinase (Ras/Raf/MEK/ERK) pathway has been documented to play a critical role in development and progression of laryngeal carcinoma [20]. For this reason, we determined whether XN was able to alter the activation level of the ERK1/2 protein. As shown in Fig. 7, a 4-h incubation of RK33 cells with various concentrations of XN resulted in a significant inhibition of ERK1/2 phosphorylation, with the most pronounced effect observed at
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Fig. 3. Effect of XN on cell cycle distribution of RK33 (A) and RK45 cells (B). Cells were incubated with different concentrations of XN for 48 h; the cell cycle distribution was assessed by flow cytometry analysis, as described in Materials and methods. All data were expressed as mean ± SD of three independent experiments. *Statistically significant at P < 0.01 in comparison to the control (untreated cells).
10 mM. In contrast, the total ERK2 expression was unchanged by the drug treatment. This study indicated that modulation of the ERK signaling cascade could be involved in the molecular mechanism of XN-mediated growth inhibition of larynx cancer cells. 4. Discussion Due to its aggressive behavior, larynx cancer is still difficult to treat with the current treatment modalities, including surgery, chemotherapy, and radiotherapy [19]. Even with new treatment protocols, no relevant improvement in 5-year overall survival rates has been observed [14]. Therefore, identifying and characterizing naturally derived compounds that have antitumor potential and display low toxicity towards normal tissues is still a challenging task. Recent in vitro and in vivo studies have demonstrated a wide range of chemopreventive and antiproliferative properties of hopderived prenylflavonoids. Among them, xanthohumol seems to be one of the most potent suppressors of malignant cells [5,35]. XN has
been described to possess activity in various types of cancer cell lines, as well as in vivo activity against human tumor xenografts in mice [10,18,32,48]. However, no data are currently available about its effectiveness with respect to laryngeal carcinoma. Our work, to the best of our knowledge, is the first to investigate in vitro the molecular effects of XN on human larynx cancer cells and to contribute to understanding its anticancer activity. In the current study, we demonstrated that XN decreased the viability of RK33 and RK45 cells; however, normal human skin fibroblasts (HSF) and rat oligodendrocytes (OLN-93) displayed relative resistance to a wide range of its concentrations. The tumor selectivity of XN observed was consistent with other data demonstrating much higher activity of XN against cancer cell lines than towards non-malignant cells [12,23,35,53]. Moreover, the low toxicity of XN and lack of a significant impact on the major organ function and metabolism, as demonstrated in toxicological studies on animals, suggest that XN might be an attractive antitumor agent [47,54].
Fig. 4. Determination of cell death in XN-treated RK33 cells. After 48 h exposure to XN (1e15 mM), the cells were stained with Hoechst 33258 and SYTOX Green and photographed with fluorescence microscopy (AeE). The bars represent the percentage of apoptotic and necrotic cells for the control and treated groups (F). Mean ± SD of three independent experiments. *Statistically significant at P < 0.01 in comparison to the control (untreated cells).
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Fig. 5. Effect of XN on the activities of caspases-3, -8, and -9 and the expression of Bcl-2. The RK33 cells were treated with XN and analyzed 48 h later by flow cytometry. The values indicate the percentage of the RK33 cells with active caspase-3 (mean ± SD of three independent experiments). *Statistically significant at P < 0.01 in comparison to the control (A). The RK33 cells were treated with XN for 48 h and then subjected to immunoblotting analysis. Equal loading was confirmed by probing for b-actin. The levels of initiator caspases and Bcl-2 are shown in (B) and (C), respectively. The intensity of the Bcl-2 bands was quantified by densitometry. The data are expressed as mean ± SD of three independent experiments. *Statistically significant at P < 0.01 in comparison to the control (untreated cells) (D).
The possible mechanism of reduced larynx tumor cell survival was related to growth inhibition, because XN reduced proliferation of both cell lines, with less potency in RK45. The differential sensitivity observed between RK33 and RK45 might indicate an XN cell-type specific effect. It is likely that the variable response of these cells to the drug treatment might be a result of distinct genetic alterations of multiple oncogenic pathways. Another possible explanation for the discrepancy of our results could lie in the differences in the growth rate and the degree of aggressiveness of the tested cells. Besides, the cytostatic/cytotoxic effect of XN was also found to be associated with interruption of the normal cell cycle dynamics.
The exposure of RK33 and RK45 lines to XN caused accumulation of cells in the G1 phase. The results were not compatible with reports of other researchers, who demonstrated cell cycle arrest in the S phase in MDA-MB435 mammary adenocarcinoma cells [22] and in PC3 prostate cancer cells [7]. However, a study by Drenzek et al. [16] on ovarian cancer cells (SKOV3, OVCAR3) revealed blockade of cell cycle progression in both S and G2/M phases. Thus, the above observations might suggest that XN is able to induce cell cycle arrest at more than one stage. Based on the increased sensitivity of the RK33 cells to the XN treatment, we further determined if the cytotoxicity observed was related to cellular apoptosis. Using fluorescence microscopy, we
Fig. 6. Effect of XN on the expression of p53, p21, and cyclin D1. The RK33 cells were treated with XN (1e15 mM) for 48 h. After the treatment, the cells were washed with PBS and extracted with protein extraction buffer. Forty micrograms of the proteins were loaded on 10% SDS-polyacrylamide gel. The expression of the proteins was analyzed by Western blot with the indicated antibodies as described in Materials and methods. Anti-b-actin antibody was used as a control for equal loading. The right panel shows representative blots; the left panel shows a densitometry analysis of the bands. All data are expressed as mean ± SD of three independent experiments. *Statistically significant at P < 0.01 in comparison to the control (untreated cells).
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Fig. 7. XN suppresses ERK1/2 activation. RK33 cells were incubated with the indicated concentrations of XN for 4 h. The cells were also treated with U0126 (a specific inhibitor of MEK1/2 kinase, 10 mM) or PMA (an ERK1/2 kinase activator, 200 nM) for 3 h to confirm inhibition or activation of ERK1/2 kinase in RK33 cells. Equal loading was confirmed by immunodetection of total ERK2. The upper panel shows representative blots; the lower panel shows a densitometry analysis of the bands. Results are expressed as mean ± SD of three independent experiments. *Statistically significant at P < 0.01 in comparison to the control (untreated cells).
found that apoptosis was the primary mode of cell death in RK33 cultures after XN administration. The most prominent increase in apoptotic cells was noticed at 10 mM of XN. In turn, the higher doses gradually diminished apoptotic cell population, but enhanced necrosis due to drug toxicity. The apoptotic effect of XN was associated with reduced expression of anti-apototic protein Bcl-2 and sequential activation of initiator caspase-9 and downstream effector caspase-3, Moreover, XN also caused degradation of procaspase-8, suggesting that both mitochondria-dependent and death receptor-dependent pathways could be involved in XNmediated cell death. Our findings were consistent with previous study by Deeb et al. [10] on prostate cancer cells. The researchers showed almost complete processing of procaspases-3, -8, and -9, activation of which was preceded by suppression of pro-survival Akt, NFkB, and mTOR signaling proteins and inhibition of Bcl-2 and survivin. The same results were obtained by Pan et al. [39]. In human glioblastoma cell lines, however, the apoptotic process was barely linked to mitochondrial damage, as evidenced by downregulation of Bcl-2, mitochondrial depolarization, and cytochrome c release [18]. Similarly, in XN-treated 3T3-L1 preadipose cells, apoptosis occurred via mitochondria-dependent events [52]. More recently, XN has also been reported to induce the caspaseindependent pathway, indicating that another form of cellular death participated in tumor killing [13]. Since XN led to the growth inhibition and apoptosis activation of larynx cancer cells, it was reasonable to determine how these effects could be achieved at the molecular levels. Therefore, we analyzed the expression of a key molecular targets including p53, p21, and cyclin D1. It is well known that wild-type p53 is a crucial tumor suppressor molecule and inductor of the apoptosis process.
Upon genomic damage or other stresses, p53 binds DNA, which in turn stimulates another gene to produce a protein called p21, which interacts with a cell division-stimulating protein (cdk2). When p21 is complexed with cdk2, the cells cannot enter the next stage of cell division [1]. In contrast to p21 and p53, cyclin D1 works as a positive regulator of cell proliferation and is essential for S phase progression [1,3]. Here, we have shown that treatment of RK33 cells with XN for 48 h causes a dose-dependent upregulation of p21 as well as p53 protein. This effect is in agreement with previous observations in K562 leukemia cells [36]. As in the study performed by Benelli et al. [4], we also found diminished expression of cyclin D1, suggesting its important function in XN-mediated G1/S phase arrest leading to RK33 cell growth inhibition. On the basis of these investigations, we postulate that modulation of p21, p53, and cyclin D1 expression might be an important mechanism by which XN acts as an antiproliferative and proapoptotic agent. There is strong evidence that abnormalities in the expression of the above proteins are frequently found in several human malignancies (including squamous cell carcinoma of the head and neck) and have been linked to tumor progression and poor prognosis [3,17,24,31,40,50,51]. Thus, the alterations of these regulatory molecules could change the aggressive behavior of cancer cells and repress their rapid and uncontrolled growth. It has been found that the ERKs signaling pathway is related to cancer cell proliferation and survival in response to multiple growth factors and some intracellular effectors [21,26]. Moreover, this cascade has been reported to be a key molecular target of several antitumor drugs (natural or synthetic origin) such as statins [45], epigallocatechin [41], resveratrol [6], sorafenib [29], and AMPA and NMDA antagonists [43,44]. These facts prompted us to evaluate the effect of XN on the activation state of ERK1/2 kinase in RK33 cells. Our results revealed a significant reduction of ERK1/2 phosphorylation, suggesting involvement of this MAPK kinase and its downstream signaling transductors in the growth suppressing potential of XN in larynx cancer cells. On the other hand, the experiments performed on glioma cells (T98G) [18] and leukemia cells (K562) [47] demonstrated strong ERK1/2 activation and another study [32] showed no change in the molecule state, indicating an opposite effect of XN to what we have presented here. The differences between our findings and those mentioned above could be cell-type specific. Nevertheless, taking into account the results of our study, we concluded that, by interfering with the MEK-ERK pathway, XN altered the expression of genes involved in cell cycle control and programmed cell death and thereby decreased the viability of RK33 cells. It remains an open question whether the cytotoxicity of XN against the larynx cancer cells might also be a result of modulation of other signal transduction cascades such as cAMP-dependent protein kinase A (cAMP/PKA) and/or the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) pathways, which are known to promote cell survival [20]. 5. Conclusions The presented studies showed for the first time that XN induced growth inhibition and apoptosis in laryngeal cancer cells. These effects were achieved by modulation of proapoptotic effectors Bcl2, caspase-8 and caspase-9, and cell cycle control molecules p21, p53, and cyclin D1. Moreover, the attenuation of survival signals ERK was probably a crucial mechanism for the anticancer action of XN in the RK33 culture. The results provide a scientific basis for using XN as a potentially effective agent for patients with laryngeal cancer, although further preclinical research is still required.
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Acknowledgments This study was supported by Maria Curie-Skłodowska University funds.
[22]
[23]
Transparency document [24]
Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.cbi.2015.08.008. [25]
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Contents lists available at ScienceDirect
Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint
Xanthohumol inhibits cell cycle progression and proliferation of larynx cancer cells in vitro ska-Brych a, *, Sylwia Katarzyna Kro l b, Adrianna Sławin ska c, Andrzej Stepulak b, ska-Graniczka b, Barbara Zdzisin Magdalena Dmoszyn a Mariusz Gagos a b c
Department of Cell Biology, Maria Curie-Sklodowska University, Akademicka 19, 20-033 Lublin, Poland Department of Biochemistry and Molecular Biology, Medical University of Lublin, Chodzki 1, 20-093 Lublin, Poland Department of Virology and Immunology, Maria Curie-Sklodowska University, Akademicka 19, 20-033 Lublin, Poland
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 April 2015 Received in revised form 19 July 2015 Accepted 11 August 2015 Available online 20 August 2015
Xanthohumol (XN), a prenylflavonoid derived from the hop plant (Humulus lupulus L.) has been found to exhibit a broad spectrum of biological properties, including anti-cancer activity. In this study, the mechanisms involved in anti-cancer activity of XN in human RK33 and RK45 larynx cancer cell lines were investigated. The effect of XN on the viability of larynx cancer and normal cells (human skin fibroblasts HSF and rat oligodendroglia-derived cells, OLN-93) was compared. Additionally, the influence of XN on proliferation, cell cycle progression, induction of apoptosis in larynx cancer cells, as well as the molecular mechanisms underlying in these processes were analyzed. XN promoted the reduction of cell viability in cancer cells, but showed low cytotoxicity to normal cells. The decrease in cell viability in the cancer cells was coupled with induction of apoptosis via two pathways. The mechanisms involved in these effects of XN were associated with cell growth inhibition by induction of cell cycle arrest in the G1 phase, increased p53 and p21/WAF1 expression levels, downregulation of cyclin D1 and Bcl-2, and activation of caspases9, -8, and -3. Moreover, this compound inhibited phosphorylation of ERK1/2, suggesting a key role of the ERKs pathway in the XN-mediated growth suppressing effects against the studied cells. These results indicate that XN could be used as a potential agent for the treatment of patients with larynx cancer. © 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Xanthohumol (XN) Larynx cancer cells Apoptosis Cell cycle arrest Extracellular signal regulated kinase 1 and 2 (ERK1/2)
1. Introduction Laryngeal squamous cell carcinoma (LSCC) is the most common malignancy of the head and neck worldwide [28]. According to the American Cancer Society, LSCC accounts for 10% of cancers in men and 4% in women [2]. Despite recent advance and achievements in diagnostic strategies and oncological treatment, LSCC is still an important therapeutic problem. The 5-year survival rates for laryngeal cancer patients with regional and metastatic LSCC demonstrated a significant decrease within last three decades, irrespective of the applied therapy [9,25]. Therefore, there is a need for discovery of novel, more effective therapeutic agents, alternative to traditionally used cisplatin (cis-diammine-dichloridoplatinum, CDDP) and 5-fluorouracil (5-FU), to improve the
* Corresponding author. skaE-mail address: [email protected] (A. Sławin Brych). http://dx.doi.org/10.1016/j.cbi.2015.08.008 0009-2797/© 2015 Elsevier Ireland Ltd. All rights reserved.
currently available treatment strategies and survival rates, particularly for patients with advanced cases of LSCC [30]. One of the most interesting and encouraging approaches is the use of natural, bioactive substances from plants as potential chemopreventive and chemotherapeutic agents in the therapy of several human cancers. There is still increasing evidence that some plant secondary metabolites could be considered as promising anticancer drugs [33,34,37,38]. Xanthohumol (XN, (E)-1-[2,4-Dihydroxy-6-methoxy-3-(3methylbut-2-enyl)phenyl]-3-(4-hydroxyphenyl)prop-2-en-1-one) is a prenylated chalcone, a representative of flavonoids, a large group of plant metabolites. XN is naturally distributed in Humulus lupulus L. (Cannabaceae) as a main component of the female inflorescences e up to 1% in dry hop cones [54]. Recently, a growing number of reports have demonstrated that XN elicits a wide spectrum of anti-cancer and chemopreventive activity such as inhibition of the metabolic activation of pro-carcinogens, induction of carcinogen-detoxifying enzymes, and inhibition of tumor growth
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in vitro [46]. XN has been shown to inhibit growth and proliferation of human breast (MCF-7) [49,54], colon (HT-29), ovarian (A2780), and prostate (DU145, PC-3) cancer cells [54,49,7] as well as Bchronic lymphocytic leukemia [32], hepatocellular carcinoma [15], and medullary thyroid cancer cells [8]. Moreover, pro-apoptotic activity of XN has also been observed in human breast cancer [47], prostate cancer [7], B-chronic lymphocytic leukemia [32], and malignant glioblastoma cells [18]. Besides these effects, it has been reported as a strong inhibitor of migration, invasion, and angiogenesis in hematological malignancies [11,36]. More importantly, this compound has additionally been shown to modulate radio- and chemosensitivity of cancer cells [4,27]. Taking into account its multiple antitumor functions and relatively nontoxic nature, XN could be a very promising drug for further in vivo investigation and potential application in oncology. The aim of the study was to assess the anti-cancer potential of XN in LSCC in vitro and mechanisms underlying its activity. To the best of our knowledge, here we report for the first time the anticancer effect of XN in LSCC.
(OLN-93) or 5 104 cells/ml (HSF) and left overnight to attach. Then, the culture medium was removed and the cells were treated with serial dilutions of XN (0.1e100 mM) in a fresh culture medium with 10% FBS or cultured in a medium alone as a control. Cell viability was determined after 48 h by the MTT assay, in which the yellow tetrazolium salt (MTT 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide) is reduced by mitochondrial dehydrogenase of viable cells to purple, insoluble crystals of formazan. The cells were incubated for 3 h with the MTT solution (5 mg/ml in PBS with Ca2þ/Mg2þ) (SigmaeAldrich) at 37 C. Then, formazan crystals were solubilized in lysing buffer (10% SDS in 0.01 N HCl) overnight at room temperature (RT). The product was quantified by measurement of absorbance at a 570 nm wavelength with the use €nnedorf, of an Infinite M200 Pro microplate reader (Tecan, Ma Switzerland) and the data were analyzed by Magellan™ Software Infinite M200 (Tecan, Switzerland). All experiments were performed in triplicate and yielded similar results.
2. Materials and methods
RK33 and RK45 cells were seeded on 96-well microplates at a density of 1000 cells/well (1 104/ml) and 3000 cells/well (3 104/ ml), respectively, and left overnight to attach. Then, the culture medium was removed and the cells were treated with serial dilutions of XN (1, 5, 10, 25, or 50 mM) in a fresh culture medium with 10% FBS. Control cells were cultured in a medium without XN. Cell proliferation was assessed after 48 h with the use of a Cell Proliferation Elisa BrdU kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's instructions. All experiments were performed in triplicate and yielded similar results.
2.1. Cell line and chemicals
2.3. Cell proliferation assessment
Human larynx cancer cells RK33 and RK45 were derived from a larynx squamous cell carcinoma taken from patients after total laryngectomy and established as stable cell lines as previously described [42]. The RK33 and RK45 cell lines were cultured in RPMI 1640 (SigmaeAldrich, St. Louis, USA) supplemented with 10% heatinactivated fetal bovine serum (FBS) (SigmaeAldrich), penicillin (100 U/ml) (SigmaeAldrich), and streptomycin (100 mg/ml) (SigmaeAldrich). Cultures were maintained at 37 C in a humidified atmosphere of 95% air and 5% CO2. The medium was replaced at 2e3-day intervals. Sub-confluent cells were usually rinsed with a phosphate-buffered solution (PBS) without Ca2þ/Mg2þ (SigmaeAldrich) and harvested with 0.25% Trypsin-EDTA (SigmaeAldrich). Human skin fibroblasts (HSF) were a laboratory strain established by an outgrowth technique from skin explants of a young person (healthy volunteer, employee of the Department of Virology and Immunology; the skin explants were obtained with the person' written informed consent). HSF were cultured in Dulbecco's modified Eagle Medium (SigmaeAldrich) containing 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin. Rat oligodendroglia-derived cells (OLN-93) were obtained from the Institute of Agricultural Medicine (Lublin, Poland) and were grown in a 1:1 mixture of Dulbecco's modified Eagle Medium and Ham's F12 Medium (SigmaeAldrich) supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin, 100 mg/ml streptomycin. The cells were maintained in a humidified incubator with 5% CO2 in air at 37 C. Xanthohumol (SigmaeAldrich) was dissolved in dimethyl sulfoxide (DMSO) and stored at 20 C until use. The final concentration of DMSO in all experiments was less than 0.01%, and all treatment conditions were compared with controls. U0126, a pharmacological inhibitor of MEK1/2 and PMA (Phorbol 12myristate 13-acetate), an activator of protein kinase C were also purchased from SigmaeAldrich.
RK33 and RK45 cells were seeded on 6-well plates (Nunc) at a density of 3 105 cells/ml and left overnight to attach. Then, the culture medium was removed and the cells were exposed to the selected dilutions of XN (0.1e10 mM) in a fresh culture medium with 10% FBS or cultured in a medium alone as a control. After 48 h, the treated cells were stained with propidium iodide (PI) using the PI/ RNase Staining Buffer (BD Biosciences, BD Pharmingen™, USA), according to the manufacturer's instructions. Briefly, the cells were harvested and centrifuged at 366 g for 5 min at RT, and the pellets were fixed in ice-cold 80% ethanol overnight at 20 C. Following fixation, the cells were centrifuged at 366 g for 5 min at 4 C, washed twice in PBS, and then incubated with 0.5 ml PI/RNase solution per 1 106 cells for 15 min in darkness at RT. Next, the stained cells were analyzed by the flow cytometry technique using a flow cytometer FACSCalibur™ (BD Biosciences, USA) equipped with a 488-nm argon-ion laser to assess the percentage of cells in phases G0/G1, S, G2/M, based on the amount of PI incorporated into DNA strains, and generate histograms of DNA distribution. The PI fluorescence intensity of individual nuclei was determined and at least 10 000 events were measured within an acquisition rate >60 events/second. Cell cycle analyses were performed with the use of non-commercial software Cylchred Version 1.0.2 for Windows (source: University of Wales, UK) and WinMDI 2.9 for Windows (source: facs.scripps.edu/software.html). All experiments were performed in triplicate and yielded similar results.
2.2. Cell viability (MTT assay)
2.5. Hoechst 33258 and SYTOX Green staining
The cancer cells and normal cells were seeded on 96-well microplates (Nunc, Langenselbold, Germany) at a density of 1 104 cells/ml (RK33), 3 104 cells/ml (RK45) and 1 104 cells/ml
RK33 cells (3 105 cells/ml) were seeded in 6-well plates and incubated with XN (1, 5, 10, or 15 mM) for 48 h. After that, the cells were detached with an Accutase solution (SigmaeAldrich) and
2.4. Cell cycle progression analysis
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harvested by centrifugation at 366 g for 5 min. The cell pellets were then washed with PBS and incubated for 15 min in a solution of Hoechst 33258 (5 mg/ml, Invitrogen, USA) and SYTOX Green (0.1 mM, Invitrogen, USA). After the incubation time, the cell suspension was placed on a microscope slide and covered with a coverslip. At least 500 cells in randomly selected microscopic fields were scored under a fluorescence microscope (Leica, Wetzlar, Germany) and categorized as normal, apoptotic, or necrotic. Live cells had a structurally normal blue nucleus. Apoptotic cells (with condensed, fragmented, or abnormally shaped nuclei) exhibited bright blue fluorescence. Cells exhibiting green fluorescent nuclei were interpreted as necrotic. 2.6. Caspase-3 activity assessment RK33 cells were seeded on 6-well plates at a density of 3 105 cells/ml and left overnight to attach. Then, the culture medium was removed and the cells were treated with serial dilutions of XN (1e15 mM) in a fresh culture medium with 10% FBS. After 48 h of the treatment, the cells were determined utilizing a PE Active Caspase-3 Apoptosis Kit (BD Biosciences, BD Pharmingen™, USA) according to the manufacturer's instructions. Briefly, the cells were harvested and washed twice with PBS, fixed and permeabilized using the Cytofix/Cytoperm Solution, then washed twice in the Perm/Wash Buffer prior to staining with rabbit monoclonal PE-conjugated anti-active caspase-3 antibody. Labeled cells were analyzed by the flow cytometry technique using a flow cytometer FACSCalibur™ equipped with a 488-nm argon-ion laser operating with CellQuest software to quantitatively assess the caspase-3 activity. All experiments were performed in triplicate and yielded similar results.
were incubated with stripping buffer (100 mM b-mercaptoethanol, 2% SDS, 62.5 mM TriseHCl, pH 6.8) for 20 min at 50 C, then washed, blocked, and incubated overnight at 4 C with primary antibodies: anti-ERK 2 or anti-b-Actin (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA). 2.8. Statistical analysis The data were presented as the mean value and standard error. Statistical analysis was performed with the one-way ANOVA with Tukey's post hoc test (GraphPad Prism5, GraphPad Software, Inc, San Diego, California, USA). Significance was accepted at P < 0.01. 3. Results 3.1. Effect of XN on the cell viability The survival rates of RK33 and RK45 cells treated with XN was assessed by an MTT assay after 48 h. As shown in Fig. 1A, the exposure to the increasing concentrations of XN (0.1e100 mM) caused a dose-dependent reduction in cell viability of both larynx cancer cell lines. However, XN was more effective for RK33 than RK45 cells. In RK33 culture, a statistically significant effect was seen at the 1 mM concentration (P < 0.01) and in RK45 cells at the 5 mM concentration (P < 0.01). Incubation with 15 mM and 25 mM induced
2.7. Protein extraction and Western blot analysis Cancer cells were seeded on 6-well plates at a density of 3 105 cells/ml and left overnight to attach. Then, the growth medium was changed and the cells were cultured with or without increasing concentrations of XN at different lengths of time (4 h for ERK1/2 activation and 48 h for p21, p53, cyclin D1, Bcl-2, and caspase-8 and caspase-9 analysis). The cells were harvested and lysed in RIPA buffer (1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 20 mM NaF, 0.5 mM DTT, 1 mM PMSF, and protease inhibitor cocktail in PBS pH 7.4) and then centrifuged at 8000 g for 10 min at 4 C. The total cellular protein concentration was quantified using the Bradford protein assay reagent (BioRad). For Western-blot analysis, supernatants of RIPA cell lysates were solubilized in 5 Laemmli Sample Buffer (50% glycerol, 10% SDS, 300 mM TriseHCl pH 6.8, 0.05% bromophenol blue, 6.25% b-mercaptoethanol) and boiled for 5 min at 100 C. Equal amounts of protein extracts were electrophoresed on 10% SDSPAGE and transferred onto a PVDF membrane (Millipore, USA). After the transfer, the membranes were blocked for 1 h at RT with 5% non-fat dry milk/TBS/0.1% Tween (SigmaeAldrich) and incubated overnight at 4 C with the following primary antibodies: antip21Waf1/Cip1, anti-p53, anti-cyclin D1, anti-Bcl-2 (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA), anti-Phospho-p44/p42 MAPK (ERK1/2) (Thr202/Tyr204) (1:2000, Cell Signaling Inc., USA), anticaspase-8, and anti-caspase-9 (1: 4000, Santa Cruz Biotechnology, Santa Cruz, CA). After incubation with the primary antibodies and washing with TBS/0.1% Tween, appropriate horseradish peroxidase-conjugated rabbit or mouse secondary antibodies (1:2000, Santa Cruz Biotechnology, Santa Cruz, CA) were added. Detection of protein bands was performed using an enhanced chemiluminescence (ECL) LUMI-LightPLUS Western Blotting Substrate Kit (Roche Diagnostics, Germany). For stripping, membranes
Fig. 1. Cytotoxic effect of XN on larynx cancer cells (A) and normal cells (B) after 48 h treatment with various concentrations of XN (0.1e100 mM). The cell viability was measured by the MTT assay. The results represent the mean ± SD of three independent experiments. *Statistically significant at P < 0.01 in comparison to the control (untreated cells).
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a decrease in the number of viable cells by 54%e71% and 46%e56% in RK33 and RK45, respectively. The highest inhibitory activity of XN was achieved at 100 mM. To study whether the XN-dependent suppression was selective for tumor cells, the impact of XN on the viability of non-malignant human skin fibroblast (HSF) and rat oligodendrocytes (OLN93) was also examined. As shown in Fig. 1B, the normal cells were less sensitive to the action of XN in comparison to the cancer cells. At the concentrations from 0.1 to 10 mM, this compound was almost non-toxic. Only high doses of XN (25e100 mM) exerted a pronounced inhibitory effect. These results indicate that XN may selectively target larynx cancer cells but spare normal cells, which is a highly attractive property of potential antitumor drugs. 3.2. XN suppresses cell proliferation and induces alteration in cell cycle distribution of larynx cancer cells RK33 and RK45 cancer cells were cultured in a complete medium in the absence (control) or presence of selected concentrations of XN for 48 h. The influence of XN on the cell proliferation was assessed by the BrdU assay. As shown in Fig. 2, XN diminished BrdU incorporation into cells in a dose-dependent manner. The XN concentrations, at which a significant inhibition in DNA synthesis was initially observed, were 1 mM for RK33 (P < 0.01) and 10 mM for RK45 cells (P < 0.01), indicating lower antiproliferative activity of XN against the RK45 cell line. The maximal dose of XN applied (50 mM) resulted in a 77% and 44% decrease in the number of proliferative cells in the RK33 and RK45 culture, respectively. Because cell cycle perturbation plays a critical role in tumorigenesis, and mitotic division, differentiation, and apoptosis are all cell cycle-dependent, we additionally analyzed the effect of XN on the cell cycle progression of both cell lines. Incubation with XN (0.1e10 mM) for 48 h led to a dose-dependent accumulation of cells in the G1 phase of the cell cycle. An increase in the G1 cell subpopulation was accompanied by cell reduction in the G2 phase. After treatment with 10 mM of XN, the percentage of cells in the G1 phase increased from 59.8% and 47.4% in the controls to 79% and 56.3% in RK33 and RK45, respectively (Fig. 3A, B). Our data suggest that the XN growth inhibitory effect could be related to inhibition of cell cycle progression.
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To further elucidate the mechanisms of the anticancer potential of XN, the more susceptible RK33 cells were used in the subsequent studies. 3.3. XN induces extrinsic and intrinsic apoptotic pathways in larynx cancer cells For determining whether the loss of cell viability observed in RK33 cultures with the presence of XN could be due to the apoptotic and/or necrotic mechanism, the cells were analyzed using double staining with Hoechst 33258 and SYTOX Green dyes. Fluorescence microscopy revealed an unchanged morphology of the nuclei in the untreated cells as well as chromatin fragmentation and appearance of apoptotic bodies in the XN-treated cells, indicating pro-apoptotic activity of this agent (Fig. 4AeF). After 48 h, at the concentrations of 1 mM, 5 mM, or 10 mM, the rate of apoptosis was higher than that of necrosis (20.7% and 10.31%, respectively at 10 mM). However, when the cells were incubated with 15 mM of XN, the drug was a stronger inducer of necrosis. At this concentration, approximately 26% of the cell population underwent necrosis, whereas 22% of the cell fraction died by apoptotic cell death (Fig. 4F). To establish whether the XN-mediated apoptosis was a caspasedependent process, we performed additional studies. The results obtained from flow cytometry clearly demonstrated that XN after 48 h caused a dose-dependent increase in the cell population positive for the presence of an active form of executor caspase-3 (Fig. 5A), reaching a maximum at 10 mM. At this concentration, the activity of caspase-3 was 6,75-fold higher than in the control cells. Moreover, XN-induced caspase-3 activity was dependent on the activation of two upstream initiator caspases, caspase-8 and caspase-9, as evidenced by a significant decrease in the procaspase8 and procaspase-9 levels and elevated expression of their active forms (Fig. 5B). Furthermore, the effect of XN on caspase-9 cleavage was correlated with reduced expression of an anti-apoptotic protein Bcl-2 (a key regulator of mitochondrial permeability and cyt c release into the cytosol); (Fig. 5C, D). Our data therefore suggest that XN may trigger apoptosis of RK33 cells by initiation of an intrinsic (caspase-9-dependent) pathway, and at least in part by extrinsic (caspase-8-dependent) apoptotic signaling. 3.4. Effect of XN on the levels of the cell cycle regulators To further study the molecular events leading to cell death and growth inhibition following XN treatment, we evaluated the expression levels of p53, p21, and cyclin D1 proteins (key molecular markers associated with the G1/S phase) in RK 33 cells using Western blot analysis. As shown in Fig. 6, after 48 h, XN (1e15 mM) affected the expression of all studied proteins in the RK33 cells. The dose of 1 mM slightly increased the expression of p53 and p21, compared to the control. Marked induction of these proteins was detected after incubation with 5 mM and 10 mM XN. In addition, XN decreased the cyclin D1 protein level and this decrease was dosedependent. A noticeable reduction of cyclin D1 expression was even seen at 1 mM. 3.5. XN inhibits ERK1/2 phosphorylation
Fig. 2. Effect of XN on cell proliferation. RK33 (A) and RK45 (B) cells were treated with increasing concentrations of XN for 48 h. Cell proliferation was determined by the BrdU incorporation assay. The results represent the mean ± SD of three independent experiments. *Statistically significant at P < 0.01 in comparison to the control (untreated cells).
Up-regulated signaling through the extracellular signalregulated kinase (Ras/Raf/MEK/ERK) pathway has been documented to play a critical role in development and progression of laryngeal carcinoma [20]. For this reason, we determined whether XN was able to alter the activation level of the ERK1/2 protein. As shown in Fig. 7, a 4-h incubation of RK33 cells with various concentrations of XN resulted in a significant inhibition of ERK1/2 phosphorylation, with the most pronounced effect observed at
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Fig. 3. Effect of XN on cell cycle distribution of RK33 (A) and RK45 cells (B). Cells were incubated with different concentrations of XN for 48 h; the cell cycle distribution was assessed by flow cytometry analysis, as described in Materials and methods. All data were expressed as mean ± SD of three independent experiments. *Statistically significant at P < 0.01 in comparison to the control (untreated cells).
10 mM. In contrast, the total ERK2 expression was unchanged by the drug treatment. This study indicated that modulation of the ERK signaling cascade could be involved in the molecular mechanism of XN-mediated growth inhibition of larynx cancer cells. 4. Discussion Due to its aggressive behavior, larynx cancer is still difficult to treat with the current treatment modalities, including surgery, chemotherapy, and radiotherapy [19]. Even with new treatment protocols, no relevant improvement in 5-year overall survival rates has been observed [14]. Therefore, identifying and characterizing naturally derived compounds that have antitumor potential and display low toxicity towards normal tissues is still a challenging task. Recent in vitro and in vivo studies have demonstrated a wide range of chemopreventive and antiproliferative properties of hopderived prenylflavonoids. Among them, xanthohumol seems to be one of the most potent suppressors of malignant cells [5,35]. XN has
been described to possess activity in various types of cancer cell lines, as well as in vivo activity against human tumor xenografts in mice [10,18,32,48]. However, no data are currently available about its effectiveness with respect to laryngeal carcinoma. Our work, to the best of our knowledge, is the first to investigate in vitro the molecular effects of XN on human larynx cancer cells and to contribute to understanding its anticancer activity. In the current study, we demonstrated that XN decreased the viability of RK33 and RK45 cells; however, normal human skin fibroblasts (HSF) and rat oligodendrocytes (OLN-93) displayed relative resistance to a wide range of its concentrations. The tumor selectivity of XN observed was consistent with other data demonstrating much higher activity of XN against cancer cell lines than towards non-malignant cells [12,23,35,53]. Moreover, the low toxicity of XN and lack of a significant impact on the major organ function and metabolism, as demonstrated in toxicological studies on animals, suggest that XN might be an attractive antitumor agent [47,54].
Fig. 4. Determination of cell death in XN-treated RK33 cells. After 48 h exposure to XN (1e15 mM), the cells were stained with Hoechst 33258 and SYTOX Green and photographed with fluorescence microscopy (AeE). The bars represent the percentage of apoptotic and necrotic cells for the control and treated groups (F). Mean ± SD of three independent experiments. *Statistically significant at P < 0.01 in comparison to the control (untreated cells).
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Fig. 5. Effect of XN on the activities of caspases-3, -8, and -9 and the expression of Bcl-2. The RK33 cells were treated with XN and analyzed 48 h later by flow cytometry. The values indicate the percentage of the RK33 cells with active caspase-3 (mean ± SD of three independent experiments). *Statistically significant at P < 0.01 in comparison to the control (A). The RK33 cells were treated with XN for 48 h and then subjected to immunoblotting analysis. Equal loading was confirmed by probing for b-actin. The levels of initiator caspases and Bcl-2 are shown in (B) and (C), respectively. The intensity of the Bcl-2 bands was quantified by densitometry. The data are expressed as mean ± SD of three independent experiments. *Statistically significant at P < 0.01 in comparison to the control (untreated cells) (D).
The possible mechanism of reduced larynx tumor cell survival was related to growth inhibition, because XN reduced proliferation of both cell lines, with less potency in RK45. The differential sensitivity observed between RK33 and RK45 might indicate an XN cell-type specific effect. It is likely that the variable response of these cells to the drug treatment might be a result of distinct genetic alterations of multiple oncogenic pathways. Another possible explanation for the discrepancy of our results could lie in the differences in the growth rate and the degree of aggressiveness of the tested cells. Besides, the cytostatic/cytotoxic effect of XN was also found to be associated with interruption of the normal cell cycle dynamics.
The exposure of RK33 and RK45 lines to XN caused accumulation of cells in the G1 phase. The results were not compatible with reports of other researchers, who demonstrated cell cycle arrest in the S phase in MDA-MB435 mammary adenocarcinoma cells [22] and in PC3 prostate cancer cells [7]. However, a study by Drenzek et al. [16] on ovarian cancer cells (SKOV3, OVCAR3) revealed blockade of cell cycle progression in both S and G2/M phases. Thus, the above observations might suggest that XN is able to induce cell cycle arrest at more than one stage. Based on the increased sensitivity of the RK33 cells to the XN treatment, we further determined if the cytotoxicity observed was related to cellular apoptosis. Using fluorescence microscopy, we
Fig. 6. Effect of XN on the expression of p53, p21, and cyclin D1. The RK33 cells were treated with XN (1e15 mM) for 48 h. After the treatment, the cells were washed with PBS and extracted with protein extraction buffer. Forty micrograms of the proteins were loaded on 10% SDS-polyacrylamide gel. The expression of the proteins was analyzed by Western blot with the indicated antibodies as described in Materials and methods. Anti-b-actin antibody was used as a control for equal loading. The right panel shows representative blots; the left panel shows a densitometry analysis of the bands. All data are expressed as mean ± SD of three independent experiments. *Statistically significant at P < 0.01 in comparison to the control (untreated cells).
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Fig. 7. XN suppresses ERK1/2 activation. RK33 cells were incubated with the indicated concentrations of XN for 4 h. The cells were also treated with U0126 (a specific inhibitor of MEK1/2 kinase, 10 mM) or PMA (an ERK1/2 kinase activator, 200 nM) for 3 h to confirm inhibition or activation of ERK1/2 kinase in RK33 cells. Equal loading was confirmed by immunodetection of total ERK2. The upper panel shows representative blots; the lower panel shows a densitometry analysis of the bands. Results are expressed as mean ± SD of three independent experiments. *Statistically significant at P < 0.01 in comparison to the control (untreated cells).
found that apoptosis was the primary mode of cell death in RK33 cultures after XN administration. The most prominent increase in apoptotic cells was noticed at 10 mM of XN. In turn, the higher doses gradually diminished apoptotic cell population, but enhanced necrosis due to drug toxicity. The apoptotic effect of XN was associated with reduced expression of anti-apototic protein Bcl-2 and sequential activation of initiator caspase-9 and downstream effector caspase-3, Moreover, XN also caused degradation of procaspase-8, suggesting that both mitochondria-dependent and death receptor-dependent pathways could be involved in XNmediated cell death. Our findings were consistent with previous study by Deeb et al. [10] on prostate cancer cells. The researchers showed almost complete processing of procaspases-3, -8, and -9, activation of which was preceded by suppression of pro-survival Akt, NFkB, and mTOR signaling proteins and inhibition of Bcl-2 and survivin. The same results were obtained by Pan et al. [39]. In human glioblastoma cell lines, however, the apoptotic process was barely linked to mitochondrial damage, as evidenced by downregulation of Bcl-2, mitochondrial depolarization, and cytochrome c release [18]. Similarly, in XN-treated 3T3-L1 preadipose cells, apoptosis occurred via mitochondria-dependent events [52]. More recently, XN has also been reported to induce the caspaseindependent pathway, indicating that another form of cellular death participated in tumor killing [13]. Since XN led to the growth inhibition and apoptosis activation of larynx cancer cells, it was reasonable to determine how these effects could be achieved at the molecular levels. Therefore, we analyzed the expression of a key molecular targets including p53, p21, and cyclin D1. It is well known that wild-type p53 is a crucial tumor suppressor molecule and inductor of the apoptosis process.
Upon genomic damage or other stresses, p53 binds DNA, which in turn stimulates another gene to produce a protein called p21, which interacts with a cell division-stimulating protein (cdk2). When p21 is complexed with cdk2, the cells cannot enter the next stage of cell division [1]. In contrast to p21 and p53, cyclin D1 works as a positive regulator of cell proliferation and is essential for S phase progression [1,3]. Here, we have shown that treatment of RK33 cells with XN for 48 h causes a dose-dependent upregulation of p21 as well as p53 protein. This effect is in agreement with previous observations in K562 leukemia cells [36]. As in the study performed by Benelli et al. [4], we also found diminished expression of cyclin D1, suggesting its important function in XN-mediated G1/S phase arrest leading to RK33 cell growth inhibition. On the basis of these investigations, we postulate that modulation of p21, p53, and cyclin D1 expression might be an important mechanism by which XN acts as an antiproliferative and proapoptotic agent. There is strong evidence that abnormalities in the expression of the above proteins are frequently found in several human malignancies (including squamous cell carcinoma of the head and neck) and have been linked to tumor progression and poor prognosis [3,17,24,31,40,50,51]. Thus, the alterations of these regulatory molecules could change the aggressive behavior of cancer cells and repress their rapid and uncontrolled growth. It has been found that the ERKs signaling pathway is related to cancer cell proliferation and survival in response to multiple growth factors and some intracellular effectors [21,26]. Moreover, this cascade has been reported to be a key molecular target of several antitumor drugs (natural or synthetic origin) such as statins [45], epigallocatechin [41], resveratrol [6], sorafenib [29], and AMPA and NMDA antagonists [43,44]. These facts prompted us to evaluate the effect of XN on the activation state of ERK1/2 kinase in RK33 cells. Our results revealed a significant reduction of ERK1/2 phosphorylation, suggesting involvement of this MAPK kinase and its downstream signaling transductors in the growth suppressing potential of XN in larynx cancer cells. On the other hand, the experiments performed on glioma cells (T98G) [18] and leukemia cells (K562) [47] demonstrated strong ERK1/2 activation and another study [32] showed no change in the molecule state, indicating an opposite effect of XN to what we have presented here. The differences between our findings and those mentioned above could be cell-type specific. Nevertheless, taking into account the results of our study, we concluded that, by interfering with the MEK-ERK pathway, XN altered the expression of genes involved in cell cycle control and programmed cell death and thereby decreased the viability of RK33 cells. It remains an open question whether the cytotoxicity of XN against the larynx cancer cells might also be a result of modulation of other signal transduction cascades such as cAMP-dependent protein kinase A (cAMP/PKA) and/or the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) pathways, which are known to promote cell survival [20]. 5. Conclusions The presented studies showed for the first time that XN induced growth inhibition and apoptosis in laryngeal cancer cells. These effects were achieved by modulation of proapoptotic effectors Bcl2, caspase-8 and caspase-9, and cell cycle control molecules p21, p53, and cyclin D1. Moreover, the attenuation of survival signals ERK was probably a crucial mechanism for the anticancer action of XN in the RK33 culture. The results provide a scientific basis for using XN as a potentially effective agent for patients with laryngeal cancer, although further preclinical research is still required.
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Acknowledgments This study was supported by Maria Curie-Skłodowska University funds.
[22]
[23]
Transparency document [24]
Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.cbi.2015.08.008. [25]
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