Silymarin causes caspases activation and apoptosis in K562 leukemia cells through inactivation of Akt pathway

Toxicology 227 (2006) 211–216

Silymarin causes caspases activation and apoptosis in K562 leukemia cells through inactivation of Akt pathway Xian Zhong a , Yongliang Zhu b , Qinghua Lu a , Jiawei Zhang a , Zhen Ge a , Shu Zheng a,∗ a

Cancer Institute, Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, Zhejiang Province, China b Department of Gastroenterology, Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, Zhejiang Province, China Received 1 June 2006; received in revised form 26 July 2006; accepted 26 July 2006 Available online 31 July 2006

Abstract Cancer is one of the largest causes of death in both men and women. Akt, overexpressed in a number of human malignancies including leukemia, is an important target in cancer prevention and/or therapy. Silymarin, a flavonoid antioxidant, has high human acceptance being used clinically for the treatment of liver diseases. In this study, Akt activity was inhibited by silymarin without changes in total Akt level associated with a prominent caspases-9 and -3 activation as well as PARP cleavage, accompanied by a strong apoptotic death and growth inhibition of K562 cells. These findings suggest that silymarin could serve as a candidate for anti-leukemia drug. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Silymarin; Akt; Apoptosis; Caspase; K562

1. Introduction Cancer is one of the largest causes of death in both men and women, claiming over 6 millions each year in the world. Constitutive activation of the Akt pathway, which is a key cause of cancer, could induce the oncogenic transformation of cells (Chung et al., 2005; Zhu et al., 2005). Akt has a wide range of downstream targets that regulate tumor-associated cell processes such as cell growth, cell cycle progression and survival (Cheng et al., 2005). Akt regulates the balance between cell survival and apoptosis through a phosphorylation

∗ Corresponding author. Tel.: +86 571 87784501; fax: +86 571 87214404. E-mail address: [email protected] (S. Zheng).

cascade that primarily alters the function of transcription factors that regulate pro- and anti-apoptotic genes (Datta et al., 1999). Evading apoptosis is a major contributor to cellular transformation, growth, and development of invasive cancer as well as drug resistance in tumors. Overexpression of Akt has been detected in a number of human malignancies (Lawlor and Alessi, 2001; Nicholson and Anderson, 2002), suggesting that Akt could be an important and a critical target in cancer prevention and/or therapy. Silymarin, isolated from the fruits of milk thistle, Silybum marianum L. Gaertn., has high human acceptance being used clinically around the world for the treatment of liver diseases (Dhiman and Chawla, 2005; Mereish et al., 1991; Saller et al., 2001). Silymarin is composed mainly of silibinin with small amounts of other silibinin stereoisomers, namely, isosilybin, dihydrosilybin,

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silydianin and silychristin (Wagner et al., 1974). A wide range of studies have shown strong cancer chemopreventive and anticancer efficacy of silymarin (or silibinin) in many kinds of tumor (Gerhauser et al., 2003; Singh and Agarwal, 2004; Tyagi et al., 2004). Studies on various animal models using different modes of administration of silymarin showed that it is non-toxic and largely free of adverse side effects in subchronic and chronic tests even at large doses and there is no known LD50 for silibinin in animal studies (Flora et al., 1998; Wellington and Jarvis, 2001). But until now, little was known about the effects of silymarin on human leukemia cells, except the study showing the differentiation effect of silibinin on HL60 cells (Kang et al., 2001; Danilenko et al., 2003). With regard to human leukemia, several studies in recent years have shown the constitutive activation of Akt (Byrd et al., 2004; Grandage et al., 2005; Tazzari et al., 2004), implicating its role in evading apoptosis and cell survival. These studies suggest that Akt could be an attractive prevention or therapeutic target in leukemia control. In the present study, we show that the polyphenolic agent silymarin inhibits the activities of Akt significantly in human chronic myeloid leukemia cell line K562, accompanied by caspases activation, apoptosis induction and proliferation inhibition.

titer plate reader (Quant Bio-Tek Instruments, Inc.). At each concentration of silymarin three different experiments were carried with three replicates. The viable cells were counted by the trypan blue exclusion assay with a hemocytometer.

2. Materials and methods

Immunoblotting was carried out following the manufacturer’s protocol. Briefly, cells were washed with PBS and lysed (50 mM HEPES, 150 mM NaCl, 1% Triton X-100, 5 mM EGTA, 50 mM ␤-glycerophosphate, 20 mM NaF, 1 mM Na3 VO4 , 2 mM phenyl-methyl sulfonyl fluoride, 10 ␮g/ml leupeptin and 10 ␮g/ml aprotinin). Cell lysates were centrifuged and the protein content was determined by Bio-Rad DC protein assay kit (Bio-Rad laboratories, Hercules, CA). Equal amounts of protein were separated by SDS-PAGE (10–15%), transferred to nitrocellulose membrane and immunoblotted with antibodies as indicated. Detection was performed using enhanced chemiluminescence (ECL) detection system. Polyclonal rabbit anti-total Akt, anti-phospho-Akt (Thr308) and anti-caspase9 antibodies were purchased from Cell Signal. Polyclonal mouse anti-cleaved caspase-3 and anti-PARP antibody were purchased from Santa Cruz, and anti-␤-actin antibody was purchased from Sigma.

2.1. Cell culture The human leukemia cell line K562, obtained from the American Type Culture Collection (Manassas, VA), was grown in an RPMI1640 containing 10% fetal bovine serum, 100 units/ml penicillin, and 100 ␮g/ml streptomycin. Cultures were maintained in a humidified atmosphere with 5% CO2 at 37 ◦ C. For treatments, exponentially growing K562 cells were collected and re-suspended in fresh culture medium. Stock solutions of silymarin (NICPBP, China, with purity > 98%) in DMSO were freshly prepared for each experiment. The final concentration of DMSO in all the cultures was 0.1%. 2.2. Cell proliferation assay Cell proliferation was assessed using the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) staining as described by Mosmann (1983). Briefly, 5 × 103 cells were incubated in 96-well plates in the presence (10, 50, 100 ␮g/ml) or absence of silymarin for 48 h in a final volume of 200 ␮l. At the end of the treatment, 40 ␮l of MTT (5 mg/ml in PBS) was added to each well and incubated for an additional 4 h at 37 ◦ C. The purple-blue MTT formazan precipitate was dissolved in 100 ␮l of DMSO. The activity of the mitochondria, reflecting cellular growth and viability, was evaluated by measuring the optical density at 570 nm on micro

2.3. Hoechst 33342 staining The K562 cells were harvested and washed with PBS, then fixed with a mixture of acetic acid–ethanol (1:3) for 10 min at room temperature, and then the cell suspension was dropped on slide glasses. After being air-dried, the cells were washed with PBS and then stained with 1 ␮g/ml Hoechst 33342 for 10 min at room temperature. The chromatin structure of the cells was examined by Zeiss fluorescence microscopy with an excitation wavelength of 340 nm and an emission wavelength of 460 nm. 2.4. Annexin V/PI staining and flow cytometry analysis K562 cells were treated with or without varying concentrations (0, 10, 50, 100 ␮g/ml) of silymarin in complete medium for 48 h. 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 (BD, San Diego, CA, USA). 2.5. Western blot analysis

3. Results 3.1. Inhibition of Akt activation without changes of total Akt level In order to investigate the effect of silymarin on Akt, various concentrations of silymarin were employed with 48 h treatment in K562 cells. As shown in Fig. 1, silymarin significantly inhibited the activation of Akt

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Fig. 2. Dose-dependent inhibition of K562 cell growth by silymarin. Cells were incubated with increasing concentration of silymarin and cell survival was determined by MTT assay. Data were mean ± S.D. of three independent experiments (** P < 0.01).

as evidences by a dose-dependent decrease in the levels of phospho-Akt proteins (Fig. 1B). Total Akt levels were then examined by immunoblotting, and no significant changes in total Akt protein levels were observed (Fig. 1A). Membrane was stripped and reprobed with ␤actin as loading control (Fig. 1). These results suggested an inhibitory effect of silymarin on Akt pathway in K562 leukemia cells.

apoptotic responses. The effect of silymarin on cell growth was then examined in K562 cells by MTT assay. The results showed that cell proliferation was inhibited in K562 cells in a dose-dependent manner (Fig. 2). Next, the morphology of silymarin treated K562 cells was investigated. Cytoplasmic condensation and cell shrinkage were visible in most K562 cells at 48 h of treatment with 100 ␮g/ml silymarin under a light microscopy (data not shown). At the same time, Hoechst 33342 staining result showed that some of the silymarin-treated cells exhibited highly condensed and fragmented nuclei morphology, which were the typical characteristics of apoptosis (Fig. 3B). In contrast, the cells in the culture without silymarin showed normal cell nuclei morphology (Fig. 3A). Apoptotic death assay employing Annexin V/PI staining followed by FACS analysis clearly showed a dose-dependent apoptotic effect of silymarin in K562 cells (Fig. 4). As shown in representative FACS analysis scatter-grams, Annexin V/PI staining of control cells showed a large viable cell population (marked as PI− AV− ) with same staining also for early apoptotic (PI− AV+ ), late apoptotic (PI+ AV+ ), and dead cells (PI+ AV− ). However, treatment of cells with silymarin at 10, 50, 100 ␮g/ml dose for 48 h resulted in a strong shift from live cells to early and late apoptotic cell population with a little change in dead cell population (Fig. 4).

3.2. Inhibition of cell proliferation and induction of apoptosis

3.3. Silymarin caused caspase-9, caspase-3, and PARP cleavages in K562 cells

Overexpression and activation of Akt is known for cell growth and survival response, which modulates many downstream effectors for survival as well as anti-

Then, we assessed the effects of silymarin on caspase activity in K562 cells. Cells were treated with different concentrations of silymarin for 48 h, and the activation

Fig. 1. Inhibitory effect of silymarin on the phosphorylation of Akt. (A) K562 cells were treated with various concentrations of silymarin (0, 10, 50 or 100 ␮g/ml) for 48 h, and then cell lysates were subjected to SDS-PAGE followed by western blotting with anti-phospho-Akt and anti-total-Akt antibodies. Signals of proteins were visualized with an ECL detection system. Each membrane was then stripped and reprobed with anti-␤-actin antibody to confirm equal protein loading. (B) Determined activities of these proteins were subsequently quantified by densitometric analysis. Data represented the mean ± S.D. of at least three independent experiments (* P < 0.05; ** P < 0.01).

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Fig. 3. Nuclei morphological changes in silymarin-treated K562 cells. K562 cells were incubated in the medium without silymarin (A) or with 100 ␮g/ml silymarin (B) for 48 h (400×). Then the cells were fixed and the nuclei were stained with Hoechst 33342. Fragmented or condensed nuclei indicative of apoptosis could be observed in the silymarin-treated group as the arrows indicated.

of caspases-9, caspases-3 and PARP was monitored by western blot analysis. It was demonstrated that caspase-9 was cleaved from 116 to 89 kDa fragment, caspase-3 was proteolytically processed into active p17 fragments, and PARP was cleaved to its characteristic 85-kDa fragment (Fig. 5).

4. Discussion In the present study, we showed that silymarin, a naturally occurring polyphenolic agent, could strongly inhibit the activity of Akt kinase without changes in total Akt protein level in K562 cells. It was also shown that

Fig. 4. Silymarin caused strong apoptotic death of K562 cells. Representative FACS analysis scatter-grams of Annexin V/PI stained control and treatment with 10, 50, 100 ␮g/ml silymarin for 48 h cells showed four different cell populations marked as: double negative (unstained) cells showing live cell population (PI− AV− ), Annexin V positive and PI negative stained cells showing early apoptosis (PI− AV+ ), Annexin V/PI double-stained cells showing late apoptosis (PI+ AV+ ), and finally PI positive and Annexin V negative stained cells showing dead cells (PI+ AV− ).

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Fig. 5. Silymarin caused caspases-9 (a) and caspases-3 (b), and PARP (c) cleavages in K562 cells. Following 48 h of cell treatments of silymarin, cells were collected and lysed in lysis buffer, and equal amounts of protein were subjected to SDS-PAGE followed by Western blotting. Membranes were probed with anti-caspase-9 (a), anti-cleaved caspase3 (b), or anti-PARP that recognizes both full and/or cleaved products (c) antibody followed by peroxidase-conjugated secondary antibody and visualization with ECL system. Each membrane was then stripped and re-probed with anti-␤-actin (d) antibody to confirm equal protein loading.

silymarin caused caspases activation and PARP cleavage as well as inhibition of cell proliferation and apoptosis induction confirmed by Hoechst 33342 staining and AV/PI staining followed by FACS analysis. The protein kinase Akt is activated in a wide variety of cancers including leukemia, and this activation results in enhanced resistance to apoptosis through multiple mechanisms (Song et al., 2005). It has been reported that human caspase-9, which acts as an initiator and an effecter of apoptosis (Donepudi and Grutter, 2002), could be phosphorylated on Ser196 by Akt/PKB, resulting in attenuation of its activity (Cardone et al., 1998). Akt is also known to regulate the antiapoptotic proteins Bcl-2 and Bcl-xl as well as the proapoptotic protein Bax (Katiyar et al., 2005; Yoo et al., 2004). Consistent with its anti-apoptotic potential by inhibiting caspases activation, we also found that a strong inhibition of Akt activity by silimarin was also associated with a strong activation of caspase-9 as evidenced by cleaved caspase products. The activation of caspase-9, in a complex known as the ‘apoptosome’ (Zou et al., 1999), is able to efficiently cleave and activate downstream executioner caspases such as caspase-3 (Rodriguez and Lazebnik, 1999). Subsequently, PARP, an intrinsic substrate for caspase-3 (Lazebnik et al., 1994), would be cleaved from 116 kDa to a typical 89 kDa fragment leading to apoptotic death. Further studies are needed in future to define the molecular mechanism of Akt activity inhibition by silymarin, and to establish the effect on animal models. The important thing to be emphasized here is the agent itself, silymarin, which is a non-toxic naturally occur-

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ring flavonoid compound widely consumed as a dietary supplement with the name ‘milk thistle extract’ for its strong anti-hepatotoxic efficacy and is also used clinically for the same purposes (Rainone, 2005; Singh and Agarwal, 2002; Singh et al., 2002a,b). There is an ongoing Phases I–II linked dose-escalation clinical trial with silibinin in prostate cancer patients, analyzing for serum silibinin levels and any associated biological response. It has been shown that up to 74 ␮g/ml total silibinin levels can be achieved in the plasma without any signs of gross toxicity in terms of diet and water consumption and weight gain profiles in silibinin-treated groups of animals versus controls (Agarwal et al., 2003). According to FDA the maximum recommended human dose of silymarin is 7 mg/kg body weight. A pilot study of oral silibinin in colorectal cancer patients has shown that silibinin level in plasma achieved 0.3–4 ␮M with repeated oral administration at dosages of 360, 720, or 1440 mg silibinin daily for 7 days (Hoh et al., 2006). Together, these results clearly indicate that the silymarin concentrations used in the present study showing strong efficacy in K562 leukemia cells are pharmacologically achievable at least in the rodent studies completed. In conclusion, the present study demonstrates that silymarin strongly inhibited Akt activity without changes in total Akt protein level in K562 cells together with caspases and PARP cleavages, induction of apoptotic death, and inhibition of cell growth. Based on these results, we conclude that silymarin is a good candidate for inhibiting Akt activity in leukemia but additional studies are needed to establish the efficacy of silymarin in leukemia cells from patients and animal cancer models, which would be useful in supporting a rationale for clinical trial in leukemia patients. Acknowledgements We thank the entire laboratory for fruitful discussions. This work was partly supported by grant from National Basic Research Program “973 Program” (G1998051200, 2004CB518707) and National Natural Science Foundation of China (30400521, 30470501). References Agarwal, C., Singh, R.P., Dhanalakshmi, S., Tyagi, A.K., Tecklenburg, M., Sclafani, R.A., Agarwal, R., 2003. 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, 8271–8282. Byrd, J.C., Stilgenbauer, S., Flinn, I.W., 2004. Chronic lymphocytic leukaemia. Hematology (Am. Soc. Hematol. Educ. Program), 163–183.

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