- Email: [email protected]
Silibinin inhibits cell growth and induces apoptosis by caspase activation, down-regulating survivin and blocking EGFR–ERK activation in renal cell carcinoma
Available online at www.sciencedirect.com
Cancer Letters 272 (2008) 61–69 www.elsevier.com/locate/canlet
Silibinin inhibits cell growth and induces apoptosis by caspase activation, down-regulating survivin and blocking EGFR–ERK activation in renal cell carcinoma Lei Li a,1, Ye Gao b,1, Linlin Zhang a, Jin Zeng a, Dalin He a,*, Yi Sun c a
Institute of Urology, The 1st Affiliated Hospital, Xi’an Jiaotong University, 277, YanTa Western Road, Xi’an, Shaanxi 710061, China b Department of Emergency, The 1st Affiliated Hospital, Xi’an Jiaotong University, Xi’an, Shaanxi 710061, China c Department of Urology, Shaanxi Provincial People’s Hospital, Shaanxi 710068, China Received 6 March 2008; received in revised form 6 March 2008; accepted 30 June 2008
Abstract Silibinin as an effective anti-cancer and chemopreventive agent in various epithelial cancer models has been reported inhibition of cancer cell growth through mitogenic signaling pathways. However, whether it could inhibit renal cell carcinoma growth and what are the underlying mechanisms is still not well elucidated. Since EGFR–MAPK and apoptosis pathways play important roles in renal cell carcinoma survival. Here, for the first time we evaluated the inhibitory proliferation effects of silibinin in renal cell carcinoma growth and examined whether silibinin modulates EGFR–MAPK and tumor apoptosis cascades signals. Our results indicated that silibinin effectively inhibits the renal cancer carcinoma Caki-1 cell proliferation and induces apoptosis through inhibiting the activation of EGFR and ERK and the expression of survivin, up-regulating the expression of p53 and triggering the cascades of caspase pathways. Our results suggested silibinin might be as one of the candidate chemopreventive agents for renal cell carcinoma therapy. Crown copyright Ó 2008 Published by Elsevier Ireland Ltd. All rights reserved. Keywords: Silibinin; Renal cell carcinoma; Apoptosis; Survivin; EGFR; ERK1/2
1. Introduction Renal cell carcinoma (RCC) is the most frequent and lethal malignant tumor of the kidney in adults. Approximately 20–30% of patients present with metastatic disease at the time of diagnosis and 20–40% of patients who undergo nephrectomy for clinically localized RCC are expected to relapse at *
1
Corresponding author. Tel.: +86 29 8532 4008. E-mail address: [email protected] (L. Li). These authors contributed equally to this work.
distant sites [1]. Once metastatic disease develops, the prognosis is extremely poor. Metastatic RCC, especially the clear cell subtype, is refractory to chemotherapy because of expression of the multidrug resistance transporters in proximal tubules [2]. Immunomodulatory therapies, of which interleukin and interferon are the major types, are no better options because of their side-effects and low response rates [2]. In additional, the modest improvement in overall survival, potential systemic toxicity and treatment related costs have also limited the utility of immunomodulatory therapeutic
0304-3835/$ - see front matter Crown copyright Ó 2008 Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2008.06.033
62
L. Li et al. / Cancer Letters 272 (2008) 61–69
reagents. Based on this concept, novel agents are rapidly developing as alternatives to targeting the unique characteristics of the RCC, specifically when this malignancy is progressed to advanced and metastatic stage. One approach to control renal cell carcinoma is growth inhibition wherein the disease is prevented, slowed, or reversed substantially by the administration of one or more non-toxic naturally occurring or synthetic agents [3,4]. Current advances in drug development have revealed cancer preventive and curative efficacies of many phytochemicals [5]. Silibinin, a naturally occurring flavonoid anti-oxidant, is isolated from milk thistle (Silybum marianum), a member of the aster family, which also includes the artichoke, a close relative [6]. Silibinin is the major active compound in silymarin, which is presented in a widely consumed dietary supplement milk thistle extract [7]. Anti-tumor efficacy of silibinin is shown in prostate, skin, colon, and bladder cancer models [8–10]. It also reported that consumption of silibinin is safe and non-toxic in animals and humans [7,8,11]. An aberrant activation of numerous signaling pathways, including both receptor and non-receptor tyrosine kinases and evasion of apoptosis, have been recognized as hallmarks of cancer cell survival and growth [12–14]. Consistent with this, several studies have shown that epidermal growth factor receptor (EGFR) and other members in their family together with the growth factors that activate them are overexpressed in both RCC tissues and derived cell lines, specifically at the advanced stage of this malignancy [15,16]. Apoptosis refers to programmed cell death in response to various intrinsic or extrinsic death signals, and is executed by series of cysteine proteases known as caspases [17,18]. Chemopreventive agents against renal cell carcinoma cells are known to exert their anti-cancer effects via modulating the expression of caspases resulting in induction of apoptosis [19–21]. These above observations suggest that targeting these molecular regulators and apoptosis pathways of cancer cell growth and survival might help in achieving a resultant growth inhibition and death of cancerous cells. Accordingly, in the present study, we investigated the efficacy and mechanisms of silibinin in human metastatic RCC Caki-1 cells. For the first time, our findings demonstrated that silibinin impart the inhibitory proliferation activity via inhibition of epidermal growth factor receptorextracellular signal regulated protein kinase
(EGFR–ERK) activation and induction of apoptosis involving modulation in caspase activation together with a decrease in survivin levels and increase p53 expression in renal cell carcinoma cells. 2. Materials and methods 2.1. Cell line and reagents Human renal cell carcinoma Caki-1 cell line was purchased from American Type Culture Collection (Manassas, VA). Cells were cultured in RPMI1640 with 10% fetal bovine serum (Gibco, NY) and 1% penicillin–streptomycin under standard culture conditions (37 °C, in humidified air containing 5% CO2). Other culture materials were purchased from Gibco (Gibco, NY). Silibinin and human recombinant EGF were purchased from Sigma Chemical Co. (St. Louis, MO). U0126 was from Alexis Biochemicals (San Diego, CA), and AG1478 was from Calbiochem (La Jolla, CA). p53 siRNA oligo was obtained from Cell Signaling Technology (Beverly, MA). Antibodies which specifically recognize phospho- and total-MAPK family members, including ERK1/2, JNK, and p38 kinase; anti-CREB, anti-cleaved caspase-3, anti-cleaved caspase-9, anti-cleaved PARP, antisurvivin, anti-phospho-EGFR, anti-EGFR, and peroxidase-conjugated secondary antibody were purchased from Cell Signaling Technology (Beverly, MA). Anti-p53 (Do-1) antibody was obtained from Santa Cruz Biotechnology. ECL system was obtained from Amersham (Piscataway, NJ). 2.2. Cell viability assay Cell viability was assessed using a tetrazoliumbased assay (MTT assay). One thousand cells in 50 ll of media per well were plated in 96-well plates in triplicate using Caki-1 cell lines. The combination treatments were added with 10 ll of 10 silibinin to give a total volume of 100 ll in each well. DMSO, in equal amounts to the treatment conditions, was added to the media in the control condition. After the different indicated time point treatment, 20 ll of the CellTiter 96Ò Aqueous One Solution (Promega, Madison, WI) was added to each well. Colorimetric analysis using a 96-well plate reader (Vmax Kinetic Microplate Reader, Molecular Devices, Sunnyvale, CA) was performed at wavelength of 490 nm. The experiments were performed in triplicate.
L. Li et al. / Cancer Letters 272 (2008) 61–69
2.3. Western immunoblot analysis Following desired treatments of Caki-1 cells with silibinin, cell lysates were prepared in non-denaturing lysis buffer (10 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 0.3 mM phenylmethylsulfonyl fluoride, 0.2 mM sodium orthovanadate, 0.5% NP-40, and 5 U/ml aprotinin). For immunoblot analyses, samples (30– 80 lg protein) were denatured in 5-sample buffer, and were subjected to SDS–PAGE on 8%, 12% or 16% Tris–glycine gels and separated proteins were transferred onto nitrocellulose membranes by Western blotting. Membranes were blocked with blocking buffer for 1 h at room temperature and probed with primary antibodies against desired molecules over night at 4 °C followed by peroxidase-conjugated appropriate secondary antibody for 1 h at room temperature and proteins were visualized by enhanced chemiluminescence detection. In each case, blots were subjected to multiple exposures on the X-ray film to ensure that the band density is in the linear range, and the bands were scanned with Adobe Photoshop 6.0 (Adobe Systems Inc., San Jose, CA).
63
dent’s t-test (SPSS 10.0 for windows). P values of less than 0.05 were considered significant. Except mentioned otherwise, all the results shown are representative of at least three independent experiments with reproducible findings. 3. Results 3.1. Silibinin inhibits growth and increases the death of human RCC Caki-1 cells Our first aim was to investigate whether silibinin treatment imparts an anti-proliferative effect against RCC
2.4. Quantitative detection of apoptosis To quantify silibinin-induced apoptotic death of Caki-1 cells, annexin V and propidium iodide (PI) staining was performed followed by flow cytometry, as described before [22]. Briefly, Caki-1 cells were plated in 60 mm dishes, and at 50% confluency, cells were treated without or with varying concentrations (0, 50, 100, and 200 lM) of silibinin in RPMI-1640 medium for different time points. In case of serum-free conditions, cells plated for overnight were switched to serum-free condition for another 24 h and then treated with silibinin under serum-free condition. At the end of each treatment, cells were collected and quantitative apoptotic death assay was performed by Annexin V and PI staining (Molecular Probes) following manufacturer’s protocol, and apoptotic cells were then analyzed immediately by flow cytometry using the FACS analysis. In apoptotic cells, Annexin V binds to phosphatidylserine, which is translocated from inner to outer leaflet of the plasma membrane. 2.5. Statistical analysis Statistical significance of differences between control and treated samples were calculated by Stu-
Fig. 1. Silibinin exhibited growth inhibition in human metastatic RCC Caki-1 cells after 24 h (A) and 48 h (B) treatment. Cells were plated in 96-well plates, treated with DMSO (control) and different concentrations of silibinin with or without serum. The cell viability was performed after 24 and 48 h treatment of silibinin as detailed in Section 2. The data shown are means ± SE of three independent plates, which were reproducible in two additional independent experiments. * means P < 0.05; ** means P < 0.01, for differences with control group.
64
L. Li et al. / Cancer Letters 272 (2008) 61–69
Caki-1 cells. As shown in Fig. 1, silibinin treatment inhibited the growth of Caki-1 cells in both dose- and timedependent manners. Silibinin treatment at 50, 100, and 200 lM doses resulted in 2–18% inhibition with serum (P < 0.05) and 4–39% inhibition without serum (P < 0.05) after 24 h of treatment (Fig. 1A) and 2–20% inhibition with serum (P < 0.05) and 20–45% (P < 0.05) growth inhibition without serum after 48 h of treatment (Fig. 1B). Much stronger cell growth inhibition was observed without serum at the 200 lM dose of silibinin following 24 h treatment and 100 lM dose of silibinin after 48 h treatment in Caki-1 cells, and cell number decreasing were probably because of the increasing of cell death (as shown in Fig. 2). However, cell growth was minimally affected by the 50 and 100 lM dose of silibinin following 24 and 48 h treatment without starvation in Caki-1 cells (Fig. 1A and 1B). Our results suggested serum might affect the inhibition effects of silibinin in Caki-1 cells, and further studies were performed under the serum-free conditions only when specially indicated in some experiments. 3.2. Silibinin causes strongly apoptotic death in Caki-1 cells The most important aspect of any given mechanistic study is its biological significance. To address this issue, we next assessed the effect of silibinin on apoptotic death in RCC Caki-1 cells under identical treatment conditions. As shown by data in Fig. 2, apoptotic death assay
employing Annexin V/PI staining followed by FACS analysis clearly showed dose- and time-dependent apoptotic effects of silibinin in Caki-1 cells. As evidenced in representative FACS analysis scatter grams, Annexin V/PI staining of control cells (0 lM silibinin) showed a large viable cell population with very fewer staining for early apoptotic, late apoptotic and dead cells. However, treatment of cells with silibinin at 50, 100, and 200 lM doses for 24 and 48 h with serum starvation condition resulted in a strong shift from live cells to early apoptotic cell populations, late apoptotic cell populations and dead cell populations. A quantitative analysis of all the data clearly demonstrated silibinin inducing apoptosis in Caki-1 cells in a dose- and time-dependent manner. Overall, compared to the control cells, silibinin treatment for 24 h at 50, 100, and 200 lM dose resulted in 4.1%, 9.4%, and 19.6% early apoptotic cells; 5.2%, 9.7%, and 13.5% of late apoptotic cells, respectively. Similar silibinin treatments for 48 h resulted in 8.8%, 13.9%, and 23.5% early apoptotic cells; 11.5%, 17.4%, and 27.1% of late apoptotic cells, respectively. However, the dead cells also increased as elevating the treatment time and the dosage of silibinin. 3.3. Silibinin inhibits ERK1/2 phosphorylation in Caki-1 cells Since we have shown that treatment of Caki-1 cells with silibinin inhibited the cell proliferation, the underly-
Fig. 2. Silibinin caused strong apoptotic death in RCC Caki-1 cells. Following 24 and 48 h of cell treatments with serum starvation detailed in Section 2. In brief, cells were collected and stained with Annexin V/PI followed by FACS analysis. Representative FACS analysis scatter-grams of Annexin V/PI stained 0, 50, 100, and 200 lM silibinin treatment showed four different cell populations marked as: double negative (unstained) cells showing live cell population (LL, lower left), Annexin V positive and PI negative stained cells showing early apoptosis (LR, lower right), Annexin V/PI double-stained cells showing late apoptosis (UR, upper right), and finally PI positive and Annexin V negative stained cells showing dead cells (UL, upper left).
L. Li et al. / Cancer Letters 272 (2008) 61–69
ing mechanisms were further investigated. Silibinin significantly inhibited the activation of ERK1/2, 100 lM dose of silibinin resulted in almost complete inhibition of phosphorylation whereas it had no significant inhibition effect on the phospho-p38, and phospho-JNK activity (Fig. 3AB). Moreover, the total amount of ERK1/2, total p38 and total JNK proteins also did not changed upon the treatment of silibinin in Caki-1 cells (data not shown). To further delineate whether the growth inhibition of Caki-1 cells upon silibinin treatment was mainly through inhibition of the ERK1/2 signaling pathway, we examined the effect of silibinin on the downstream effectors of the ERK1/2 signaling pathway. The cAMP response element-binding (CREB), one of the downstream target gene transactivated by phosphorylation of ERK1/2, was analyzed by immunoblotting. The results indicated that those CREB expression was strongly decreased at 100 lM dose and nearly completely inhibited at 200 lM dose in response to silibinin (Fig. 3C).
Fig. 3. An inhibitory effect of silibinin on the phosphorylation of ERK1/2. Caki-1 cells were treated with the indicated doses of silibinin (0, 50, 100, and 200 lM) for 24 or/and 48 h, and then cell lysates were subjected to SDS–PAGE followed by Western blotting. As described in Section 2. The membranes were probed with anti-phospho-ERK1/2, anti-phospho-p38, anti-phosphoJNK, anti-total-ERK1/2 antibodies after 24 h (A) and 48 h (B) silibinin treatment, respectively. The ERK1/2 downstream effectors CREB also probed with anti-CREB at 24 h treatment of silibinin to confirm the ERK1/2 pathways inhibition (C).
65
3.4. Silibinin causes caspase-9 and -3, and PARP cleavages, p53 up-regulation and down-regulates survivin expression in Caki-1 cells In order to assess the molecular mechanism of silibinin on the apoptosis in Caki-1 cells, various concentrations and time points of silibinin treatments were employed, followed by Western blotting detection of the activation of the apoptosis related protein expression. As shown in Fig. 4A, silibinin treatment in a dose-dependent manner for 48 h resulted in an increase of the caspase-9 activation of the cleaved product of procaspase-9. Moreover, similar treatments at 200 lM dose caused very strong caspase-9 activation at both 24 and 48 h. The ultimate step in the caspase cascade is the activation of caspase-3, which results in PARP cleavage [23]. We next assessed whether there was a similar effect on caspase-3 activation. As shown in Fig. 4A, consistent with caspase-9 observations, silibinin treatment both for 24 and 48 h at 200 lM dose resulted in a very strong caspase-3 activation, which presented in a dose-dependent activation. Similar observations were also evidenced in terms of PARP cleavage.
Fig. 4. Silibinin activated the cascades of caspases up-regulated p53 and down-regulated survivin protein expression in Caki-1 cells. Following 24 and 48 h of cell treatments detailed in Section 2, cells were collected and lysed and then subjected to SDS– PAGE followed by Western blotting. Membranes were probed with anti-cleaved caspase-9, anti-cleaved caspase-3 or anti-PARP antibody (A) and apoptosis inhibited protein anti-survivin (B). p53 protein was detected following 100 lM silibinin treatment for 24 h. The p53 siRNA oligo transfection was performed according the manufacturer’s protocol (C).
66
L. Li et al. / Cancer Letters 272 (2008) 61–69
The major anti-apoptotic function of survivin, one of the members of inhibitors of apoptosis (IAP), has been established via its interaction with caspases, resulting in a block of apoptotic cell death [24]. In addition, p53 protein is also involved in RCC cell apoptosis regulation [25]. Based on our findings that silibinin causes up-regulation of the cleavage caspase proteins, we therefore assessed whether silibinin treatment also resulted in the expression change of p53 and survivin. As shown in Fig. 4B and C, silibinin treatment of cells resulted in a strong to complete decrease in survivin protein levels and increase the expression of p53. The treatment of cells at 50 and 100 lM doses did not show any effect on survivin levels, but 100 lM silibinin treatment for 24 h resulted in apparently decrease in survivin protein levels in Caki-1 cells. A complete down-regulation in survivin protein was observed following 200 lM dose treatment at both 24 and 48 h. One hundred micro molar silibinin treatment for 24 h also resulted in increase of p53 protein levelsin Caki-1 cells. Inhibition of p53 expression could increase the expression of survi-
vin which was abolished by adding 100 lM silibinin in Caki-1 cells. Our results indicated p53 are probably involved in silibinin mediated surviving down-regulation. 3.5. Silibinin inhibits ERK1/2 phosphorylation through inhibition of EGFR signaling in Caki-1 cells Previous studies showed silibinin could inhibit growth factor receptor mediated mitogenic and cell survival signaling in tumor cells [23,26], we therefore assessed the effect of silibinin on the inhibition of ERK through EGFR signaling. As is shown in Fig. 5A and C, after 24 h of serum starvation in the absence of EGF, we only see a slight activation of EGFR and ERK1/2 in Caki-1 cells, indicating this starvation time was sufficient to diminish EGFR signaling in this cells. EGF (100 ng/ml) treatments were used for activating EGFR in the silibinin, AG1478, or U0126 treated cells according previous study [27]. AG1478 (200 nM), a specific pharmacological inhibitor of EGFR activation, and U0126 (10 lM), a specific pharmacological inhibitor of ERK activation, were used
Fig. 5. Silibinin inhibited ERK1/2 phosphorylation through inhibition of EGFR signaling in Caki-1 cells. Serum-starved Caki-1 cells were treated with varying concentrations of either silibinin, or AG1478, a specific inhibitor of EGFR activation, or U0126, a specific inhibitor of ERK activation for 2 h. EGFR (A) and ERK1/2 (C) activation was then assessed following addition of EGF, or negative control without EGF, or positive control with U0126 or AG1478. Detection of immunoblotted proteins shows that silibinin diminished phosphorylation of EGFR and ERK1/2 in a dose-dependent manner. Silibinin diminishes EGF-induced cell proliferation, also measured by MTT assays as mentioned in Section 2. Serum-starved cells were treated with varying concentrations of silibinin (0–200 lM) with for 2 h, followed by addition of EGF to 100 ng/ml. Cell proliferation was determined 48 h later with AG1478 (B) or with U0126 (D). Numbers shown are with standard deviation from triplicate samples.
L. Li et al. / Cancer Letters 272 (2008) 61–69
as a positive control. As seen in Fig. 5A and C, AG1478 or U0126 could decrease the EGF-induced tyrosine phosphorylation of EGFR or ERK in Caki-1 cells as expected. Silibinin at 200 lM significantly decreased the EGFinduced tyrosine phosphorylation of EGFR and ERK. These results confirm that inhibition of EGFR activation by silibinin might be an essential mechanism in ERK activation in Caki-1 cells. Since silibinin might inhibit the activation of EGFR and ERK by EGF stimulation in Caki-1 cells, we following observed the Caki-1 proliferation effects upon the inhibition of the EGFR–ERK activation by silibinin. As shown in Fig. 5B and 5D, cells were treated with EGF and varying amounts of silibinin. The proliferation of Caki-1 was stimulated approximately 35% by EGF. However, the addition of silibinin along with EGF to Caki-1 cells inhibited EGF activation, and caused decreased cell proliferation in a dose-dependent manner. The proliferation was inhibited by specific inhibitor of EGFR and ERK, as a positive control, which could inhibit 23% and 27%, respectively. Proliferation was inhibited by 30– 36%, 77–79%, and 18–80% along with 50, 100, and 200 lM silibinin and 100 ng/ml EGF treatment in Caki1 cells, respectively. The difference between proliferation in the presence of 50, 100 or 200 lM silibinin compared to no silibinin (all with EGF added), was significant in Caki-1 cells.
4. Discussion RCC is the most frequent and lethal malignant tumor of the kidney in adults. Because of the unique characteristics of RCC, developing novel non-toxic natural alternative therapeutic reagents have been become an important research field for many oncologist. An aberrant activation of numerous growth signaling pathways and evasion of apoptosis have been recognized as hallmarks of cancer cell survival and growth including RCC [12–14]. Silibinin has been reported inhibits many cancer cells growth via activating mitogenic pathways and induces apoptosis [28–31]. In our present study, we firstly demonstrated silibinin exerts the potent inhibitory proliferation effects through both dosage- and time-dependent manners in metastatic RCC Caki-1 cells, and the inhibition of Caki-1 cells proliferation mediated by silibinin is via inhibition of the activation of EGFR–ERK pathways but not through inhibition of the activation of p38 mitogen-activated protein kinases and stress-activated protein kinase/c-JUN NH2-terminal kinase 1/2 pathways, which is partially in agreement of the previous studies that silibinin inhibit cancer cells growth through
67
phosphorylation of ERK1/2, p-JNK, and p38 but not inhibited Akt phosphorylation [31–34]. It also reported oral silibinin inhibited lung tumor growth and reduced systemic toxicity of doxorubicin with an enhanced therapeutic efficacy most likely via an inhibition of doxorubicin induced chemoresistance involving NFjB signaling [35]. In renal cell carcinoma, silibinin could suppress renal cell carcinoma SN12K1 cell growth in vitro, which were independent of insulin-like growth factor binding protein 3 (IGFBP-3) pathways [36]. However, Cheung et al. also reported oral administration of silibinin suppressed local and metastatic tumor growth in vivo in an orthotopic xenograft model of RCC and this anti-neoplastic action of silibinin might involve IGFBP-3 [37]. Moreover, Qi et al . demonstrated that EGFR is involved in silibinin-induced cytotoxicity in 9 L rat glioma cell line [27]. Base on this point, we then studied whether EGFR activation also involved in silibinin-induced inhibitory proliferation effects in RCC Caki-1 cells. Our results support the hypothesis that silibinin anti-proliferation to RCC Caki-1 cancer cells were involved in the EGFR signaling pathway. The findings presented here provide a rationale for understanding the growth inhibition effect of silibinin in Caki-1 cells. Numerous previous studies demonstrated silibinin could induce in many cancer cells apoptosis but very few report in RCC cells [30,38]. In present studies we firstly displayed that silibinin might induce Caki-1 cells apoptosis in both time and dosage dependent manners via activation of caspase pathways, inhibition of survivin expression and up-regulation of p53 expression. Survivin, as the inhibitor of apoptosis (IAP) molecule, plays an important role in human RCC neoplasia [39]. In this regard, several molecular epidemiological studies have concluded that there is a strong association between increased survivin levels and progression of human RCC [40–42]. It has been reported that silibinin could inhibit the expression of survivn in human bladder cancer cells in vitro and in human prostate carcinoma PC-3 tumor xenograft [29,43]. It also showed that silibinin modulated CDKICDK-cyclin cascade and activated caspase-3 causing growth inhibition and apoptotic death in human bladder cancer cells [28]. Very recent study indicated silibinin inhibited constitutively active Stat3 and induced apoptosis in prostate cancer DU145 cells, suggesting this might have potential significance in therapeutic intervention of this deadly malignancy [44]. Consistent with these observations, our results
68
L. Li et al. / Cancer Letters 272 (2008) 61–69
provides a strong rationale for future studies evaluating preventive and/or intervention strategies for silibinin in RCC pre-clinical models. In conclusion, the results of the present studies showed that silibinin causes a strong inhibition of the activation of EGFR–ERK pathways, up-regulation of p53 expression and down-regulation of survivin expression in human RCC Caki-1 cells together with activation of caspases and PARP cleavages, induction of apoptotic death, and inhibition of cell growth, and additional studies are needed to establish the efficacy of silibinin in pre-clinical RCC models, which would be useful in supporting a rationale for clinical trial in renal cell carcinoma patients. Acknowledgements We thank Celine Billard for her technical assistances and Dr. Kaihua Zhao for helpful suggestions of review this paper. References [1] N.K. Janzen, H.L. Kim, R.A. Figlin, A.S. Belldegrun, Surveillance after radical or partial nephrectomy for localized renal cell carcinoma and management of recurrent disease, Urol. Clin. North Am. 30 (2003) 843–852. [2] H.T. Cohen, F.J. McGovern, Renal-cell carcinoma, N. Engl. J. Med. 353 (2005) 2477–2490. [3] A. Bex, M. Kerst, H. Mallo, W. Meinhardt, S. Horenblas, G.C. de Gast, Interferon-a 2b as medical selection for nephrectomy in patients with synchronous metastatic renal cell carcinoma: a consecutive study, Eur. Urol. 49 (2006) 76– 81. [4] A.M. Kamat, D.L. Lamm, Chemoprevention of bladder cancer, Urol. Clin. North Am. 29 (2002) 157–168. [5] Y.J. Surh, Cancer chemoprevention with dietary phytochemicals, Nat. Rev. Cancer 3 (2003) 768–780. [6] R.P. Singh, R. Agarwal, Flavonoid antioxidant silymarin and skin cancer, Antioxid. Redox Signal. 4 (2002) 655–663. [7] K. Wellington, B. Jarvis, Silymarin: a review of its clinical properties in the management of hepatic disorders, Biodrugs 15 (2001) 465–489. [8] R.P. Singh, S. Dhanalakshmi, A.K. Tyagi, D.C. Chan, C. Agarwal, R. Agarwal, Dietary feeding of silibinin inhibits advance human prostate carcinoma growth in athymic nude mice and increases plasma insulin-like growth factor-binding protein-3 levels, Cancer Res. 62 (2002) 3063–3069. [9] S.K. Katiyar, N.J. Korman, H. Mukhtar, R. Agarwal, Protective effects of silymarin against photocarcinogenesis in a mouse skin model, J. Natl. Cancer Inst. 89 (1997) 556–566. [10] H. Kohno, T. Tanaka, K. Kawabata, Y. Hirose, S. Sugie, H. Tsuda, H. Mori, Silymarin, a naturally occurring polyphenolic antioxidant flavonoid, inhibits azoxymethane-induced colon carcinogenesis in male F344 rats, Int. J. Cancer 101 (2002) 461–468.
[11] C. Agarwal, R.P. Singh, S. Dhanalakshmi, A.K. Tyagi, M. Tecklenburg, R.A. Sclafani, R. Agarwal, Silibinin upregulates the expression of cyclin-dependent kinase inhibitors and causes cell cycle arrest and apoptosis in human colon carcinoma HT-29 cells, Oncogene 22 (2003) 8271–8282. [12] M. Eto, S. Naito, Molecular targeting therapy for renal cell carcinoma, Int. J. Clin. Oncol. 11 (2006) 209–213. [13] J.M. Larkin, T. Eisen, Kinase inhibitors in the treatment of renal cell carcinoma, Crit. Rev. Oncol. Hematol. 60 (2006) 216–226. [14] M. Staehler, K. Rohrmann, N. Haseke, C.G. Stief, M. Siebels, Targeted agents for the treatment of advanced renal cell carcinoma, Curr. Drug Targets 6 (2005) 835–846. [15] D. Cohen, B. Lane, T. Jin, C. Magi-Galluzzi, J. Finke, B.I. Rini, R.M. Bukowski, M. Zhou, The prognostic significance of epidermal growth factor receptor expression in clear-cell renal cell carcinoma: a call for standardized methods for immunohistochemical evaluation, Clin. Genitourin. Cancer 5 (2007) 264–270. [16] G. Badalian, K. Derecskei, A. Szendroi, M. Szendroi, J. Timar, EGFR and VEGFR2 protein expressions in bone metastases of clear cell renal cancer, Anticancer Res. 27 (2007) 889–894. [17] D.W. Nicholson, N.A. Thornberry, Caspases: killer proteases, Trends Biochem. Sci. 22 (1997) 299–306. [18] B. Fadeel, S. Orrenius, Apoptosis: a basic biological phenomenon with wide-ranging implications in human disease, J. Intern. Med. 258 (2005) 479–517. [19] T.J. Lee, E.J. Kim, S. Kim, E.M. Jung, J.W. Park, S.H. Jeong, S.E. Park, Y.H. Yoo, T.K. Kwon, Caspase-dependent and caspase-independent apoptosis induced by evodiamine in human leukemic U937 cells, Mol. Cancer Ther. 5 (2006) 2398–2407. [20] R. Srinivasan, W.M. Linehan, Targeted for destruction: the molecular basis for development of novel therapeutic strategies in renal cell cancer, J. Clin. Oncol. 23 (2005) 410–412. [21] X.X. Wu, O. Ogawa, Y. Kakehi, Enhancement of arsenic trioxide-induced apoptosis in renal cell carcinoma cells by Lbuthionine sulfoximine, Int. J. Oncol. 24 (2004) 1489–1497. [22] R.P. Singh, S. Dhanalakshmi, S. Mohan, C. Agarwal, R. Agarwal, Silibinin inhibits UVB- and epidermal growth factor-induced mitogenic and cell survival signaling involving activator protein-1 and nuclear factor-jB in mouse epidermal JB6 cells, Mol. Cancer Ther. 5 (2006) 1145–1153. [23] S.W. Lowe, A.W. Lin, Apoptosis in cancer, Carcinogenesis 21 (2000) 485–495. [24] D.C. Altieri, Validating survivin as a cancer therapeutic target, Nat. Rev. Cancer 3 (2003) 46–54. [25] G. Li, C. Lambert, A. Gentil-Perret, C. Genin, J. Tostain, Molecular and cytometric analysis of renal cell carcinoma cells. Concepts, techniques and prospects, Prog. Urol. 13 (2003) 1–13. [26] X. Zi, J. Zhang, R. Agarwal, M. Pollak, Silibinin upregulates insulin-like growth factor-binding protein 3 expression and inhibits proliferation of androgen-independent prostate cancer cells, Cancer Res. 60 (2000) 5617–5620. [27] L. Qi, R.P. Singh, Y. Lu, R. Agarwal, G.S. Harrison, A. Franzusoff, L.M. Glode, Epidermal growth factor receptor mediates silibinin-induced cytotoxicity in a rat glioma cell line, Cancer Biol. Ther. 2 (2003) 526–531. [28] A. Tyagi, C. Agarwal, G. Harrison, L.M. Glode, R. Agarwal, Silibinin causes cell cycle arrest and apoptosis in
L. Li et al. / Cancer Letters 272 (2008) 61–69
[29]
[30]
[31]
[32]
[33]
[34]
[35]
human bladder transitional cell carcinoma cells by regulating CDKI-CDK-cyclin cascade, and caspase 3 and PARP cleavages, Carcinogenesis 25 (2004) 1711–1720. A.K. Tyagi, C. Agarwal, R.P. Singh, K.R. Shroyer, L.M. Glode, R. Agarwal, Silibinin down-regulates survivin protein and mRNA expression and causes caspases activation and apoptosis in human bladder transitional-cell papilloma RT4 cells, Biochem. Biophys. Res. Commun. 312 (2003) 1178– 1184. R.P. Singh, R. Agarwal, Prostate cancer chemoprevention by silibinin: bench to bedside, Mol. Carcinog. 45 (2006) 436– 442. P.N. Chen, Y.S. Hsieh, C.L. Chiang, H.L. Chiou, S.F. Yang, S.C. Chu, Silibinin inhibits invasion of oral cancer cells by suppressing the MAPK pathway, J. Dent. Res. 85 (2006) 220–225. P.N. Chen, Y.S. Hsieh, H.L. Chiou, S.C. Chu, Silibinin inhibits cell invasion through inactivation of both PI3K-Akt and MAPK signaling pathways, Chem. Biol. Interact. 156 (2005) 141–150. S. Mohan, S. Dhanalakshmi, G.U. Mallikarjuna, R.P. Singh, R. Agarwal, Silibinin modulates UVB-induced apoptosis via mitochondrial proteins, caspases activation, and mitogen-activated protein kinase signaling in human epidermoid carcinoma A431 cells, Biochem. Biophys. Res. Commun. 320 (2004) 183–189. R.P. Singh, A.K. Tyagi, J. Zhao, R. Agarwal, Silymarin inhibits growth and causes regression of established skin tumors in SENCAR mice via modulation of mitogenactivated protein kinases and induction of apoptosis, Carcinogenesis 23 (2002) 499–510. R.P. Singh, G.U. Mallikarjuna, G. Sharma, S. Dhanalakshmi, A.K. Tyagi, D.C. Chan, C. Agarwal, R. Agarwal, Oral silibinin inhibits lung tumor growth in athymic nude mice and forms a novel chemocombination with doxorubicin targeting nuclear factor jB-mediated inducible chemoresistance, Clin. Cancer Res. 10 (2004) 8641–8647.
69
[36] C.W. Cheung, D.A. Vesey, D.L. Nicol, D.W. Johnson, Silibinin inhibits renal cell carcinoma via mechanisms that are independent of insulin-like growth factor-binding protein 3, BJU Int. 99 (2007) 454–460. [37] C.W. Cheung, P.J. Taylor, C.M. Kirkpatrick, D.A. Vesey, G.C. Gobe, C. Winterford, D.L. Nicol, D.W. Johnson, Therapeutic value of orally administered silibinin in renal cell carcinoma: manipulation of insulin-like growth factor binding protein-3 levels, BJU Int. 100 (2007) 438–444. [38] R.P. Singh, R. Agarwal, Mechanisms and preclinical efficacy of silibinin in preventing skin cancer, Eur. J. Cancer 41 (2005) 1969–1979. [39] C. Mahotka, M. Wenzel, E. Springer, H.E. Gabbert, C.D. Gerharz, Survivin-deltaEx3 and survivin-2B: two novel splice variants of the apoptosis inhibitor survivin with different antiapoptotic properties, Cancer Res. 59 (1999) 6097–6102. [40] B. Ljungberg, Prognostic markers in renal cell carcinoma, Curr. Opin. Urol. 17 (2007) 303–308. [41] A.E. Krambeck, H. Dong, R.H. Thompson, S.M. Kuntz, C.M. Lohse, B.C. Leibovich, M.L. Blute, T.J. Sebo, J.C. Cheville, A.S. Parker, E.D. Kwon, Survivin and b7-h1 are collaborative predictors of survival and represent potential therapeutic targets for patients with renal cell carcinoma, Clin. Cancer Res. 13 (2007) 1749–1756. [42] S.S. Byun, W.G. Yeo, S.E. Lee, E. Lee, Expression of survivin in renal cell carcinomas: association with pathologic features and clinical outcome, Urology 69 (2007) 34–37. [43] M.C. Comelli, U. Mengs, C. Schneider, M. Prosdocimi, Toward the definition of the mechanism of action of silymarin: activities related to cellular protection from toxic damage induced by chemotherapy, Integr. Cancer Ther. 6 (2007) 120–129. [44] C. Agarwal, A. Tyagi, M. Kaur, R. Agarwal, Silibinin inhibits constitutive activation of Stat3 and causes caspase activation and apoptotic death of human prostate carcinoma DU145 cells, Carcinogenesis 28 (2007) 1463–1470.
Cancer Letters 272 (2008) 61–69 www.elsevier.com/locate/canlet
Silibinin inhibits cell growth and induces apoptosis by caspase activation, down-regulating survivin and blocking EGFR–ERK activation in renal cell carcinoma Lei Li a,1, Ye Gao b,1, Linlin Zhang a, Jin Zeng a, Dalin He a,*, Yi Sun c a
Institute of Urology, The 1st Affiliated Hospital, Xi’an Jiaotong University, 277, YanTa Western Road, Xi’an, Shaanxi 710061, China b Department of Emergency, The 1st Affiliated Hospital, Xi’an Jiaotong University, Xi’an, Shaanxi 710061, China c Department of Urology, Shaanxi Provincial People’s Hospital, Shaanxi 710068, China Received 6 March 2008; received in revised form 6 March 2008; accepted 30 June 2008
Abstract Silibinin as an effective anti-cancer and chemopreventive agent in various epithelial cancer models has been reported inhibition of cancer cell growth through mitogenic signaling pathways. However, whether it could inhibit renal cell carcinoma growth and what are the underlying mechanisms is still not well elucidated. Since EGFR–MAPK and apoptosis pathways play important roles in renal cell carcinoma survival. Here, for the first time we evaluated the inhibitory proliferation effects of silibinin in renal cell carcinoma growth and examined whether silibinin modulates EGFR–MAPK and tumor apoptosis cascades signals. Our results indicated that silibinin effectively inhibits the renal cancer carcinoma Caki-1 cell proliferation and induces apoptosis through inhibiting the activation of EGFR and ERK and the expression of survivin, up-regulating the expression of p53 and triggering the cascades of caspase pathways. Our results suggested silibinin might be as one of the candidate chemopreventive agents for renal cell carcinoma therapy. Crown copyright Ó 2008 Published by Elsevier Ireland Ltd. All rights reserved. Keywords: Silibinin; Renal cell carcinoma; Apoptosis; Survivin; EGFR; ERK1/2
1. Introduction Renal cell carcinoma (RCC) is the most frequent and lethal malignant tumor of the kidney in adults. Approximately 20–30% of patients present with metastatic disease at the time of diagnosis and 20–40% of patients who undergo nephrectomy for clinically localized RCC are expected to relapse at *
1
Corresponding author. Tel.: +86 29 8532 4008. E-mail address: [email protected] (L. Li). These authors contributed equally to this work.
distant sites [1]. Once metastatic disease develops, the prognosis is extremely poor. Metastatic RCC, especially the clear cell subtype, is refractory to chemotherapy because of expression of the multidrug resistance transporters in proximal tubules [2]. Immunomodulatory therapies, of which interleukin and interferon are the major types, are no better options because of their side-effects and low response rates [2]. In additional, the modest improvement in overall survival, potential systemic toxicity and treatment related costs have also limited the utility of immunomodulatory therapeutic
0304-3835/$ - see front matter Crown copyright Ó 2008 Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2008.06.033
62
L. Li et al. / Cancer Letters 272 (2008) 61–69
reagents. Based on this concept, novel agents are rapidly developing as alternatives to targeting the unique characteristics of the RCC, specifically when this malignancy is progressed to advanced and metastatic stage. One approach to control renal cell carcinoma is growth inhibition wherein the disease is prevented, slowed, or reversed substantially by the administration of one or more non-toxic naturally occurring or synthetic agents [3,4]. Current advances in drug development have revealed cancer preventive and curative efficacies of many phytochemicals [5]. Silibinin, a naturally occurring flavonoid anti-oxidant, is isolated from milk thistle (Silybum marianum), a member of the aster family, which also includes the artichoke, a close relative [6]. Silibinin is the major active compound in silymarin, which is presented in a widely consumed dietary supplement milk thistle extract [7]. Anti-tumor efficacy of silibinin is shown in prostate, skin, colon, and bladder cancer models [8–10]. It also reported that consumption of silibinin is safe and non-toxic in animals and humans [7,8,11]. An aberrant activation of numerous signaling pathways, including both receptor and non-receptor tyrosine kinases and evasion of apoptosis, have been recognized as hallmarks of cancer cell survival and growth [12–14]. Consistent with this, several studies have shown that epidermal growth factor receptor (EGFR) and other members in their family together with the growth factors that activate them are overexpressed in both RCC tissues and derived cell lines, specifically at the advanced stage of this malignancy [15,16]. Apoptosis refers to programmed cell death in response to various intrinsic or extrinsic death signals, and is executed by series of cysteine proteases known as caspases [17,18]. Chemopreventive agents against renal cell carcinoma cells are known to exert their anti-cancer effects via modulating the expression of caspases resulting in induction of apoptosis [19–21]. These above observations suggest that targeting these molecular regulators and apoptosis pathways of cancer cell growth and survival might help in achieving a resultant growth inhibition and death of cancerous cells. Accordingly, in the present study, we investigated the efficacy and mechanisms of silibinin in human metastatic RCC Caki-1 cells. For the first time, our findings demonstrated that silibinin impart the inhibitory proliferation activity via inhibition of epidermal growth factor receptorextracellular signal regulated protein kinase
(EGFR–ERK) activation and induction of apoptosis involving modulation in caspase activation together with a decrease in survivin levels and increase p53 expression in renal cell carcinoma cells. 2. Materials and methods 2.1. Cell line and reagents Human renal cell carcinoma Caki-1 cell line was purchased from American Type Culture Collection (Manassas, VA). Cells were cultured in RPMI1640 with 10% fetal bovine serum (Gibco, NY) and 1% penicillin–streptomycin under standard culture conditions (37 °C, in humidified air containing 5% CO2). Other culture materials were purchased from Gibco (Gibco, NY). Silibinin and human recombinant EGF were purchased from Sigma Chemical Co. (St. Louis, MO). U0126 was from Alexis Biochemicals (San Diego, CA), and AG1478 was from Calbiochem (La Jolla, CA). p53 siRNA oligo was obtained from Cell Signaling Technology (Beverly, MA). Antibodies which specifically recognize phospho- and total-MAPK family members, including ERK1/2, JNK, and p38 kinase; anti-CREB, anti-cleaved caspase-3, anti-cleaved caspase-9, anti-cleaved PARP, antisurvivin, anti-phospho-EGFR, anti-EGFR, and peroxidase-conjugated secondary antibody were purchased from Cell Signaling Technology (Beverly, MA). Anti-p53 (Do-1) antibody was obtained from Santa Cruz Biotechnology. ECL system was obtained from Amersham (Piscataway, NJ). 2.2. Cell viability assay Cell viability was assessed using a tetrazoliumbased assay (MTT assay). One thousand cells in 50 ll of media per well were plated in 96-well plates in triplicate using Caki-1 cell lines. The combination treatments were added with 10 ll of 10 silibinin to give a total volume of 100 ll in each well. DMSO, in equal amounts to the treatment conditions, was added to the media in the control condition. After the different indicated time point treatment, 20 ll of the CellTiter 96Ò Aqueous One Solution (Promega, Madison, WI) was added to each well. Colorimetric analysis using a 96-well plate reader (Vmax Kinetic Microplate Reader, Molecular Devices, Sunnyvale, CA) was performed at wavelength of 490 nm. The experiments were performed in triplicate.
L. Li et al. / Cancer Letters 272 (2008) 61–69
2.3. Western immunoblot analysis Following desired treatments of Caki-1 cells with silibinin, cell lysates were prepared in non-denaturing lysis buffer (10 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 0.3 mM phenylmethylsulfonyl fluoride, 0.2 mM sodium orthovanadate, 0.5% NP-40, and 5 U/ml aprotinin). For immunoblot analyses, samples (30– 80 lg protein) were denatured in 5-sample buffer, and were subjected to SDS–PAGE on 8%, 12% or 16% Tris–glycine gels and separated proteins were transferred onto nitrocellulose membranes by Western blotting. Membranes were blocked with blocking buffer for 1 h at room temperature and probed with primary antibodies against desired molecules over night at 4 °C followed by peroxidase-conjugated appropriate secondary antibody for 1 h at room temperature and proteins were visualized by enhanced chemiluminescence detection. In each case, blots were subjected to multiple exposures on the X-ray film to ensure that the band density is in the linear range, and the bands were scanned with Adobe Photoshop 6.0 (Adobe Systems Inc., San Jose, CA).
63
dent’s t-test (SPSS 10.0 for windows). P values of less than 0.05 were considered significant. Except mentioned otherwise, all the results shown are representative of at least three independent experiments with reproducible findings. 3. Results 3.1. Silibinin inhibits growth and increases the death of human RCC Caki-1 cells Our first aim was to investigate whether silibinin treatment imparts an anti-proliferative effect against RCC
2.4. Quantitative detection of apoptosis To quantify silibinin-induced apoptotic death of Caki-1 cells, annexin V and propidium iodide (PI) staining was performed followed by flow cytometry, as described before [22]. Briefly, Caki-1 cells were plated in 60 mm dishes, and at 50% confluency, cells were treated without or with varying concentrations (0, 50, 100, and 200 lM) of silibinin in RPMI-1640 medium for different time points. In case of serum-free conditions, cells plated for overnight were switched to serum-free condition for another 24 h and then treated with silibinin under serum-free condition. At the end of each treatment, cells were collected and quantitative apoptotic death assay was performed by Annexin V and PI staining (Molecular Probes) following manufacturer’s protocol, and apoptotic cells were then analyzed immediately by flow cytometry using the FACS analysis. In apoptotic cells, Annexin V binds to phosphatidylserine, which is translocated from inner to outer leaflet of the plasma membrane. 2.5. Statistical analysis Statistical significance of differences between control and treated samples were calculated by Stu-
Fig. 1. Silibinin exhibited growth inhibition in human metastatic RCC Caki-1 cells after 24 h (A) and 48 h (B) treatment. Cells were plated in 96-well plates, treated with DMSO (control) and different concentrations of silibinin with or without serum. The cell viability was performed after 24 and 48 h treatment of silibinin as detailed in Section 2. The data shown are means ± SE of three independent plates, which were reproducible in two additional independent experiments. * means P < 0.05; ** means P < 0.01, for differences with control group.
64
L. Li et al. / Cancer Letters 272 (2008) 61–69
Caki-1 cells. As shown in Fig. 1, silibinin treatment inhibited the growth of Caki-1 cells in both dose- and timedependent manners. Silibinin treatment at 50, 100, and 200 lM doses resulted in 2–18% inhibition with serum (P < 0.05) and 4–39% inhibition without serum (P < 0.05) after 24 h of treatment (Fig. 1A) and 2–20% inhibition with serum (P < 0.05) and 20–45% (P < 0.05) growth inhibition without serum after 48 h of treatment (Fig. 1B). Much stronger cell growth inhibition was observed without serum at the 200 lM dose of silibinin following 24 h treatment and 100 lM dose of silibinin after 48 h treatment in Caki-1 cells, and cell number decreasing were probably because of the increasing of cell death (as shown in Fig. 2). However, cell growth was minimally affected by the 50 and 100 lM dose of silibinin following 24 and 48 h treatment without starvation in Caki-1 cells (Fig. 1A and 1B). Our results suggested serum might affect the inhibition effects of silibinin in Caki-1 cells, and further studies were performed under the serum-free conditions only when specially indicated in some experiments. 3.2. Silibinin causes strongly apoptotic death in Caki-1 cells The most important aspect of any given mechanistic study is its biological significance. To address this issue, we next assessed the effect of silibinin on apoptotic death in RCC Caki-1 cells under identical treatment conditions. As shown by data in Fig. 2, apoptotic death assay
employing Annexin V/PI staining followed by FACS analysis clearly showed dose- and time-dependent apoptotic effects of silibinin in Caki-1 cells. As evidenced in representative FACS analysis scatter grams, Annexin V/PI staining of control cells (0 lM silibinin) showed a large viable cell population with very fewer staining for early apoptotic, late apoptotic and dead cells. However, treatment of cells with silibinin at 50, 100, and 200 lM doses for 24 and 48 h with serum starvation condition resulted in a strong shift from live cells to early apoptotic cell populations, late apoptotic cell populations and dead cell populations. A quantitative analysis of all the data clearly demonstrated silibinin inducing apoptosis in Caki-1 cells in a dose- and time-dependent manner. Overall, compared to the control cells, silibinin treatment for 24 h at 50, 100, and 200 lM dose resulted in 4.1%, 9.4%, and 19.6% early apoptotic cells; 5.2%, 9.7%, and 13.5% of late apoptotic cells, respectively. Similar silibinin treatments for 48 h resulted in 8.8%, 13.9%, and 23.5% early apoptotic cells; 11.5%, 17.4%, and 27.1% of late apoptotic cells, respectively. However, the dead cells also increased as elevating the treatment time and the dosage of silibinin. 3.3. Silibinin inhibits ERK1/2 phosphorylation in Caki-1 cells Since we have shown that treatment of Caki-1 cells with silibinin inhibited the cell proliferation, the underly-
Fig. 2. Silibinin caused strong apoptotic death in RCC Caki-1 cells. Following 24 and 48 h of cell treatments with serum starvation detailed in Section 2. In brief, cells were collected and stained with Annexin V/PI followed by FACS analysis. Representative FACS analysis scatter-grams of Annexin V/PI stained 0, 50, 100, and 200 lM silibinin treatment showed four different cell populations marked as: double negative (unstained) cells showing live cell population (LL, lower left), Annexin V positive and PI negative stained cells showing early apoptosis (LR, lower right), Annexin V/PI double-stained cells showing late apoptosis (UR, upper right), and finally PI positive and Annexin V negative stained cells showing dead cells (UL, upper left).
L. Li et al. / Cancer Letters 272 (2008) 61–69
ing mechanisms were further investigated. Silibinin significantly inhibited the activation of ERK1/2, 100 lM dose of silibinin resulted in almost complete inhibition of phosphorylation whereas it had no significant inhibition effect on the phospho-p38, and phospho-JNK activity (Fig. 3AB). Moreover, the total amount of ERK1/2, total p38 and total JNK proteins also did not changed upon the treatment of silibinin in Caki-1 cells (data not shown). To further delineate whether the growth inhibition of Caki-1 cells upon silibinin treatment was mainly through inhibition of the ERK1/2 signaling pathway, we examined the effect of silibinin on the downstream effectors of the ERK1/2 signaling pathway. The cAMP response element-binding (CREB), one of the downstream target gene transactivated by phosphorylation of ERK1/2, was analyzed by immunoblotting. The results indicated that those CREB expression was strongly decreased at 100 lM dose and nearly completely inhibited at 200 lM dose in response to silibinin (Fig. 3C).
Fig. 3. An inhibitory effect of silibinin on the phosphorylation of ERK1/2. Caki-1 cells were treated with the indicated doses of silibinin (0, 50, 100, and 200 lM) for 24 or/and 48 h, and then cell lysates were subjected to SDS–PAGE followed by Western blotting. As described in Section 2. The membranes were probed with anti-phospho-ERK1/2, anti-phospho-p38, anti-phosphoJNK, anti-total-ERK1/2 antibodies after 24 h (A) and 48 h (B) silibinin treatment, respectively. The ERK1/2 downstream effectors CREB also probed with anti-CREB at 24 h treatment of silibinin to confirm the ERK1/2 pathways inhibition (C).
65
3.4. Silibinin causes caspase-9 and -3, and PARP cleavages, p53 up-regulation and down-regulates survivin expression in Caki-1 cells In order to assess the molecular mechanism of silibinin on the apoptosis in Caki-1 cells, various concentrations and time points of silibinin treatments were employed, followed by Western blotting detection of the activation of the apoptosis related protein expression. As shown in Fig. 4A, silibinin treatment in a dose-dependent manner for 48 h resulted in an increase of the caspase-9 activation of the cleaved product of procaspase-9. Moreover, similar treatments at 200 lM dose caused very strong caspase-9 activation at both 24 and 48 h. The ultimate step in the caspase cascade is the activation of caspase-3, which results in PARP cleavage [23]. We next assessed whether there was a similar effect on caspase-3 activation. As shown in Fig. 4A, consistent with caspase-9 observations, silibinin treatment both for 24 and 48 h at 200 lM dose resulted in a very strong caspase-3 activation, which presented in a dose-dependent activation. Similar observations were also evidenced in terms of PARP cleavage.
Fig. 4. Silibinin activated the cascades of caspases up-regulated p53 and down-regulated survivin protein expression in Caki-1 cells. Following 24 and 48 h of cell treatments detailed in Section 2, cells were collected and lysed and then subjected to SDS– PAGE followed by Western blotting. Membranes were probed with anti-cleaved caspase-9, anti-cleaved caspase-3 or anti-PARP antibody (A) and apoptosis inhibited protein anti-survivin (B). p53 protein was detected following 100 lM silibinin treatment for 24 h. The p53 siRNA oligo transfection was performed according the manufacturer’s protocol (C).
66
L. Li et al. / Cancer Letters 272 (2008) 61–69
The major anti-apoptotic function of survivin, one of the members of inhibitors of apoptosis (IAP), has been established via its interaction with caspases, resulting in a block of apoptotic cell death [24]. In addition, p53 protein is also involved in RCC cell apoptosis regulation [25]. Based on our findings that silibinin causes up-regulation of the cleavage caspase proteins, we therefore assessed whether silibinin treatment also resulted in the expression change of p53 and survivin. As shown in Fig. 4B and C, silibinin treatment of cells resulted in a strong to complete decrease in survivin protein levels and increase the expression of p53. The treatment of cells at 50 and 100 lM doses did not show any effect on survivin levels, but 100 lM silibinin treatment for 24 h resulted in apparently decrease in survivin protein levels in Caki-1 cells. A complete down-regulation in survivin protein was observed following 200 lM dose treatment at both 24 and 48 h. One hundred micro molar silibinin treatment for 24 h also resulted in increase of p53 protein levelsin Caki-1 cells. Inhibition of p53 expression could increase the expression of survi-
vin which was abolished by adding 100 lM silibinin in Caki-1 cells. Our results indicated p53 are probably involved in silibinin mediated surviving down-regulation. 3.5. Silibinin inhibits ERK1/2 phosphorylation through inhibition of EGFR signaling in Caki-1 cells Previous studies showed silibinin could inhibit growth factor receptor mediated mitogenic and cell survival signaling in tumor cells [23,26], we therefore assessed the effect of silibinin on the inhibition of ERK through EGFR signaling. As is shown in Fig. 5A and C, after 24 h of serum starvation in the absence of EGF, we only see a slight activation of EGFR and ERK1/2 in Caki-1 cells, indicating this starvation time was sufficient to diminish EGFR signaling in this cells. EGF (100 ng/ml) treatments were used for activating EGFR in the silibinin, AG1478, or U0126 treated cells according previous study [27]. AG1478 (200 nM), a specific pharmacological inhibitor of EGFR activation, and U0126 (10 lM), a specific pharmacological inhibitor of ERK activation, were used
Fig. 5. Silibinin inhibited ERK1/2 phosphorylation through inhibition of EGFR signaling in Caki-1 cells. Serum-starved Caki-1 cells were treated with varying concentrations of either silibinin, or AG1478, a specific inhibitor of EGFR activation, or U0126, a specific inhibitor of ERK activation for 2 h. EGFR (A) and ERK1/2 (C) activation was then assessed following addition of EGF, or negative control without EGF, or positive control with U0126 or AG1478. Detection of immunoblotted proteins shows that silibinin diminished phosphorylation of EGFR and ERK1/2 in a dose-dependent manner. Silibinin diminishes EGF-induced cell proliferation, also measured by MTT assays as mentioned in Section 2. Serum-starved cells were treated with varying concentrations of silibinin (0–200 lM) with for 2 h, followed by addition of EGF to 100 ng/ml. Cell proliferation was determined 48 h later with AG1478 (B) or with U0126 (D). Numbers shown are with standard deviation from triplicate samples.
L. Li et al. / Cancer Letters 272 (2008) 61–69
as a positive control. As seen in Fig. 5A and C, AG1478 or U0126 could decrease the EGF-induced tyrosine phosphorylation of EGFR or ERK in Caki-1 cells as expected. Silibinin at 200 lM significantly decreased the EGFinduced tyrosine phosphorylation of EGFR and ERK. These results confirm that inhibition of EGFR activation by silibinin might be an essential mechanism in ERK activation in Caki-1 cells. Since silibinin might inhibit the activation of EGFR and ERK by EGF stimulation in Caki-1 cells, we following observed the Caki-1 proliferation effects upon the inhibition of the EGFR–ERK activation by silibinin. As shown in Fig. 5B and 5D, cells were treated with EGF and varying amounts of silibinin. The proliferation of Caki-1 was stimulated approximately 35% by EGF. However, the addition of silibinin along with EGF to Caki-1 cells inhibited EGF activation, and caused decreased cell proliferation in a dose-dependent manner. The proliferation was inhibited by specific inhibitor of EGFR and ERK, as a positive control, which could inhibit 23% and 27%, respectively. Proliferation was inhibited by 30– 36%, 77–79%, and 18–80% along with 50, 100, and 200 lM silibinin and 100 ng/ml EGF treatment in Caki1 cells, respectively. The difference between proliferation in the presence of 50, 100 or 200 lM silibinin compared to no silibinin (all with EGF added), was significant in Caki-1 cells.
4. Discussion RCC is the most frequent and lethal malignant tumor of the kidney in adults. Because of the unique characteristics of RCC, developing novel non-toxic natural alternative therapeutic reagents have been become an important research field for many oncologist. An aberrant activation of numerous growth signaling pathways and evasion of apoptosis have been recognized as hallmarks of cancer cell survival and growth including RCC [12–14]. Silibinin has been reported inhibits many cancer cells growth via activating mitogenic pathways and induces apoptosis [28–31]. In our present study, we firstly demonstrated silibinin exerts the potent inhibitory proliferation effects through both dosage- and time-dependent manners in metastatic RCC Caki-1 cells, and the inhibition of Caki-1 cells proliferation mediated by silibinin is via inhibition of the activation of EGFR–ERK pathways but not through inhibition of the activation of p38 mitogen-activated protein kinases and stress-activated protein kinase/c-JUN NH2-terminal kinase 1/2 pathways, which is partially in agreement of the previous studies that silibinin inhibit cancer cells growth through
67
phosphorylation of ERK1/2, p-JNK, and p38 but not inhibited Akt phosphorylation [31–34]. It also reported oral silibinin inhibited lung tumor growth and reduced systemic toxicity of doxorubicin with an enhanced therapeutic efficacy most likely via an inhibition of doxorubicin induced chemoresistance involving NFjB signaling [35]. In renal cell carcinoma, silibinin could suppress renal cell carcinoma SN12K1 cell growth in vitro, which were independent of insulin-like growth factor binding protein 3 (IGFBP-3) pathways [36]. However, Cheung et al. also reported oral administration of silibinin suppressed local and metastatic tumor growth in vivo in an orthotopic xenograft model of RCC and this anti-neoplastic action of silibinin might involve IGFBP-3 [37]. Moreover, Qi et al . demonstrated that EGFR is involved in silibinin-induced cytotoxicity in 9 L rat glioma cell line [27]. Base on this point, we then studied whether EGFR activation also involved in silibinin-induced inhibitory proliferation effects in RCC Caki-1 cells. Our results support the hypothesis that silibinin anti-proliferation to RCC Caki-1 cancer cells were involved in the EGFR signaling pathway. The findings presented here provide a rationale for understanding the growth inhibition effect of silibinin in Caki-1 cells. Numerous previous studies demonstrated silibinin could induce in many cancer cells apoptosis but very few report in RCC cells [30,38]. In present studies we firstly displayed that silibinin might induce Caki-1 cells apoptosis in both time and dosage dependent manners via activation of caspase pathways, inhibition of survivin expression and up-regulation of p53 expression. Survivin, as the inhibitor of apoptosis (IAP) molecule, plays an important role in human RCC neoplasia [39]. In this regard, several molecular epidemiological studies have concluded that there is a strong association between increased survivin levels and progression of human RCC [40–42]. It has been reported that silibinin could inhibit the expression of survivn in human bladder cancer cells in vitro and in human prostate carcinoma PC-3 tumor xenograft [29,43]. It also showed that silibinin modulated CDKICDK-cyclin cascade and activated caspase-3 causing growth inhibition and apoptotic death in human bladder cancer cells [28]. Very recent study indicated silibinin inhibited constitutively active Stat3 and induced apoptosis in prostate cancer DU145 cells, suggesting this might have potential significance in therapeutic intervention of this deadly malignancy [44]. Consistent with these observations, our results
68
L. Li et al. / Cancer Letters 272 (2008) 61–69
provides a strong rationale for future studies evaluating preventive and/or intervention strategies for silibinin in RCC pre-clinical models. In conclusion, the results of the present studies showed that silibinin causes a strong inhibition of the activation of EGFR–ERK pathways, up-regulation of p53 expression and down-regulation of survivin expression in human RCC Caki-1 cells together with activation of caspases and PARP cleavages, induction of apoptotic death, and inhibition of cell growth, and additional studies are needed to establish the efficacy of silibinin in pre-clinical RCC models, which would be useful in supporting a rationale for clinical trial in renal cell carcinoma patients. Acknowledgements We thank Celine Billard for her technical assistances and Dr. Kaihua Zhao for helpful suggestions of review this paper. References [1] N.K. Janzen, H.L. Kim, R.A. Figlin, A.S. Belldegrun, Surveillance after radical or partial nephrectomy for localized renal cell carcinoma and management of recurrent disease, Urol. Clin. North Am. 30 (2003) 843–852. [2] H.T. Cohen, F.J. McGovern, Renal-cell carcinoma, N. Engl. J. Med. 353 (2005) 2477–2490. [3] A. Bex, M. Kerst, H. Mallo, W. Meinhardt, S. Horenblas, G.C. de Gast, Interferon-a 2b as medical selection for nephrectomy in patients with synchronous metastatic renal cell carcinoma: a consecutive study, Eur. Urol. 49 (2006) 76– 81. [4] A.M. Kamat, D.L. Lamm, Chemoprevention of bladder cancer, Urol. Clin. North Am. 29 (2002) 157–168. [5] Y.J. Surh, Cancer chemoprevention with dietary phytochemicals, Nat. Rev. Cancer 3 (2003) 768–780. [6] R.P. Singh, R. Agarwal, Flavonoid antioxidant silymarin and skin cancer, Antioxid. Redox Signal. 4 (2002) 655–663. [7] K. Wellington, B. Jarvis, Silymarin: a review of its clinical properties in the management of hepatic disorders, Biodrugs 15 (2001) 465–489. [8] R.P. Singh, S. Dhanalakshmi, A.K. Tyagi, D.C. Chan, C. Agarwal, R. Agarwal, Dietary feeding of silibinin inhibits advance human prostate carcinoma growth in athymic nude mice and increases plasma insulin-like growth factor-binding protein-3 levels, Cancer Res. 62 (2002) 3063–3069. [9] S.K. Katiyar, N.J. Korman, H. Mukhtar, R. Agarwal, Protective effects of silymarin against photocarcinogenesis in a mouse skin model, J. Natl. Cancer Inst. 89 (1997) 556–566. [10] H. Kohno, T. Tanaka, K. Kawabata, Y. Hirose, S. Sugie, H. Tsuda, H. Mori, Silymarin, a naturally occurring polyphenolic antioxidant flavonoid, inhibits azoxymethane-induced colon carcinogenesis in male F344 rats, Int. J. Cancer 101 (2002) 461–468.
[11] C. Agarwal, R.P. Singh, S. Dhanalakshmi, A.K. Tyagi, M. Tecklenburg, R.A. Sclafani, R. Agarwal, Silibinin upregulates the expression of cyclin-dependent kinase inhibitors and causes cell cycle arrest and apoptosis in human colon carcinoma HT-29 cells, Oncogene 22 (2003) 8271–8282. [12] M. Eto, S. Naito, Molecular targeting therapy for renal cell carcinoma, Int. J. Clin. Oncol. 11 (2006) 209–213. [13] J.M. Larkin, T. Eisen, Kinase inhibitors in the treatment of renal cell carcinoma, Crit. Rev. Oncol. Hematol. 60 (2006) 216–226. [14] M. Staehler, K. Rohrmann, N. Haseke, C.G. Stief, M. Siebels, Targeted agents for the treatment of advanced renal cell carcinoma, Curr. Drug Targets 6 (2005) 835–846. [15] D. Cohen, B. Lane, T. Jin, C. Magi-Galluzzi, J. Finke, B.I. Rini, R.M. Bukowski, M. Zhou, The prognostic significance of epidermal growth factor receptor expression in clear-cell renal cell carcinoma: a call for standardized methods for immunohistochemical evaluation, Clin. Genitourin. Cancer 5 (2007) 264–270. [16] G. Badalian, K. Derecskei, A. Szendroi, M. Szendroi, J. Timar, EGFR and VEGFR2 protein expressions in bone metastases of clear cell renal cancer, Anticancer Res. 27 (2007) 889–894. [17] D.W. Nicholson, N.A. Thornberry, Caspases: killer proteases, Trends Biochem. Sci. 22 (1997) 299–306. [18] B. Fadeel, S. Orrenius, Apoptosis: a basic biological phenomenon with wide-ranging implications in human disease, J. Intern. Med. 258 (2005) 479–517. [19] T.J. Lee, E.J. Kim, S. Kim, E.M. Jung, J.W. Park, S.H. Jeong, S.E. Park, Y.H. Yoo, T.K. Kwon, Caspase-dependent and caspase-independent apoptosis induced by evodiamine in human leukemic U937 cells, Mol. Cancer Ther. 5 (2006) 2398–2407. [20] R. Srinivasan, W.M. Linehan, Targeted for destruction: the molecular basis for development of novel therapeutic strategies in renal cell cancer, J. Clin. Oncol. 23 (2005) 410–412. [21] X.X. Wu, O. Ogawa, Y. Kakehi, Enhancement of arsenic trioxide-induced apoptosis in renal cell carcinoma cells by Lbuthionine sulfoximine, Int. J. Oncol. 24 (2004) 1489–1497. [22] R.P. Singh, S. Dhanalakshmi, S. Mohan, C. Agarwal, R. Agarwal, Silibinin inhibits UVB- and epidermal growth factor-induced mitogenic and cell survival signaling involving activator protein-1 and nuclear factor-jB in mouse epidermal JB6 cells, Mol. Cancer Ther. 5 (2006) 1145–1153. [23] S.W. Lowe, A.W. Lin, Apoptosis in cancer, Carcinogenesis 21 (2000) 485–495. [24] D.C. Altieri, Validating survivin as a cancer therapeutic target, Nat. Rev. Cancer 3 (2003) 46–54. [25] G. Li, C. Lambert, A. Gentil-Perret, C. Genin, J. Tostain, Molecular and cytometric analysis of renal cell carcinoma cells. Concepts, techniques and prospects, Prog. Urol. 13 (2003) 1–13. [26] X. Zi, J. Zhang, R. Agarwal, M. Pollak, Silibinin upregulates insulin-like growth factor-binding protein 3 expression and inhibits proliferation of androgen-independent prostate cancer cells, Cancer Res. 60 (2000) 5617–5620. [27] L. Qi, R.P. Singh, Y. Lu, R. Agarwal, G.S. Harrison, A. Franzusoff, L.M. Glode, Epidermal growth factor receptor mediates silibinin-induced cytotoxicity in a rat glioma cell line, Cancer Biol. Ther. 2 (2003) 526–531. [28] A. Tyagi, C. Agarwal, G. Harrison, L.M. Glode, R. Agarwal, Silibinin causes cell cycle arrest and apoptosis in
L. Li et al. / Cancer Letters 272 (2008) 61–69
[29]
[30]
[31]
[32]
[33]
[34]
[35]
human bladder transitional cell carcinoma cells by regulating CDKI-CDK-cyclin cascade, and caspase 3 and PARP cleavages, Carcinogenesis 25 (2004) 1711–1720. A.K. Tyagi, C. Agarwal, R.P. Singh, K.R. Shroyer, L.M. Glode, R. Agarwal, Silibinin down-regulates survivin protein and mRNA expression and causes caspases activation and apoptosis in human bladder transitional-cell papilloma RT4 cells, Biochem. Biophys. Res. Commun. 312 (2003) 1178– 1184. R.P. Singh, R. Agarwal, Prostate cancer chemoprevention by silibinin: bench to bedside, Mol. Carcinog. 45 (2006) 436– 442. P.N. Chen, Y.S. Hsieh, C.L. Chiang, H.L. Chiou, S.F. Yang, S.C. Chu, Silibinin inhibits invasion of oral cancer cells by suppressing the MAPK pathway, J. Dent. Res. 85 (2006) 220–225. P.N. Chen, Y.S. Hsieh, H.L. Chiou, S.C. Chu, Silibinin inhibits cell invasion through inactivation of both PI3K-Akt and MAPK signaling pathways, Chem. Biol. Interact. 156 (2005) 141–150. S. Mohan, S. Dhanalakshmi, G.U. Mallikarjuna, R.P. Singh, R. Agarwal, Silibinin modulates UVB-induced apoptosis via mitochondrial proteins, caspases activation, and mitogen-activated protein kinase signaling in human epidermoid carcinoma A431 cells, Biochem. Biophys. Res. Commun. 320 (2004) 183–189. R.P. Singh, A.K. Tyagi, J. Zhao, R. Agarwal, Silymarin inhibits growth and causes regression of established skin tumors in SENCAR mice via modulation of mitogenactivated protein kinases and induction of apoptosis, Carcinogenesis 23 (2002) 499–510. R.P. Singh, G.U. Mallikarjuna, G. Sharma, S. Dhanalakshmi, A.K. Tyagi, D.C. Chan, C. Agarwal, R. Agarwal, Oral silibinin inhibits lung tumor growth in athymic nude mice and forms a novel chemocombination with doxorubicin targeting nuclear factor jB-mediated inducible chemoresistance, Clin. Cancer Res. 10 (2004) 8641–8647.
69
[36] C.W. Cheung, D.A. Vesey, D.L. Nicol, D.W. Johnson, Silibinin inhibits renal cell carcinoma via mechanisms that are independent of insulin-like growth factor-binding protein 3, BJU Int. 99 (2007) 454–460. [37] C.W. Cheung, P.J. Taylor, C.M. Kirkpatrick, D.A. Vesey, G.C. Gobe, C. Winterford, D.L. Nicol, D.W. Johnson, Therapeutic value of orally administered silibinin in renal cell carcinoma: manipulation of insulin-like growth factor binding protein-3 levels, BJU Int. 100 (2007) 438–444. [38] R.P. Singh, R. Agarwal, Mechanisms and preclinical efficacy of silibinin in preventing skin cancer, Eur. J. Cancer 41 (2005) 1969–1979. [39] C. Mahotka, M. Wenzel, E. Springer, H.E. Gabbert, C.D. Gerharz, Survivin-deltaEx3 and survivin-2B: two novel splice variants of the apoptosis inhibitor survivin with different antiapoptotic properties, Cancer Res. 59 (1999) 6097–6102. [40] B. Ljungberg, Prognostic markers in renal cell carcinoma, Curr. Opin. Urol. 17 (2007) 303–308. [41] A.E. Krambeck, H. Dong, R.H. Thompson, S.M. Kuntz, C.M. Lohse, B.C. Leibovich, M.L. Blute, T.J. Sebo, J.C. Cheville, A.S. Parker, E.D. Kwon, Survivin and b7-h1 are collaborative predictors of survival and represent potential therapeutic targets for patients with renal cell carcinoma, Clin. Cancer Res. 13 (2007) 1749–1756. [42] S.S. Byun, W.G. Yeo, S.E. Lee, E. Lee, Expression of survivin in renal cell carcinomas: association with pathologic features and clinical outcome, Urology 69 (2007) 34–37. [43] M.C. Comelli, U. Mengs, C. Schneider, M. Prosdocimi, Toward the definition of the mechanism of action of silymarin: activities related to cellular protection from toxic damage induced by chemotherapy, Integr. Cancer Ther. 6 (2007) 120–129. [44] C. Agarwal, A. Tyagi, M. Kaur, R. Agarwal, Silibinin inhibits constitutive activation of Stat3 and causes caspase activation and apoptotic death of human prostate carcinoma DU145 cells, Carcinogenesis 28 (2007) 1463–1470.