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Diallyl trisulfide induces Bcl-2 and caspase-3-dependent apoptosis via downregulation of Akt phosphorylation in human T24 bladder cancer cells.

ABSTRACT

It is well known that the garlic-derived organosulfur compounds (OSCs) are effective to inhibit a variety of human cancers such as prostate, breast, colon, skin, lung, and bladder cancers. Herein, the pro-apoptotic effects of diallyl trisulfide (DATS), one of garlic-derived OSCs, on T24 bladder cancer cells were investigated. The results demonstrated that DATS suppressed proliferation of T24 bladder cancer cells in a dose- and time-dependent manner which was associated with induced G2/M Phase cell cycle arrest and apoptosis. Mechanistically, DATS inhibits phosphatidylinositol 3'-kinase/Akt activation that, in turn, results in modulation of Bcl-2 family proteins, leading to enhanced apoptosis of T24 cells. These findings suggest that DATS may be an effective way for treating human bladder and other types of cancers.

[c] 2009 Elsevier GmbH. All rights reserved.

ARTICLE INFO

Keywords:

Bladder cancer

Akt

Diallyl trisulfide

Cell cycle arrest

Apoptosis

Introduction

It was estimated that there were 67 160 new cases of bladder cancer diagnosed in the United States in 2007 (Jemal et al. 2007). Bladder cancer is the fourth most common cancer in men after prostate, lung, and colorectal cancers. Despite recent advances in surgical and chemotherapeutic procedures, the 5-year survival rate in patients with invasive and metastatic bladder cancer remains very low. Therefore, the development of other therapeutic approaches is needed to prolong the survival and improve the quality of life in these patients.

Chemoprevention is regarded as one of the most promising and realistic approaches in the prevention of human cancer. Among naturally occurring products, garlic-derived organosulfur compounds (OSCs) represents important and promising chemo-preventive agents. Recently, several epidemiologic investigations have shown that the risk of various types of cancers is inversely related to intake of garlic (Allium sativum) (You et al. 1989; Hsing et al. 2002; Challier et al. 1998).For example, a population-based case-control study conducted in China reported that men consuming >10g/day of total Allium vegetables had a significantly lower risk of prostate cancer than those with low total Allium vegetable intake (<2.2g/day) (Hsing et al. 2002).

These epidemiologic observations are supported by laborator-ial and experimental animal studies which provide convincing evidences that garlic and some of its associated organosulfur components are effective to inhibit a variety of cancers and cancer cells in culture, including prostate, breast, colon, skin, uterine, esophagus, lung, and bladder cancers (Herman-Antosiewicz and Singh 2004; Milner 2001; Lau et al. 1986). For example, it was shown that diallyl trisulfide (DATS) can trigger apoptosis in PC-3 and DU145 human prostate cancer cells through inhibiting the expression of Bcl-2 protein, and activating ERK1/2 and JNK pathways, as well as inactivating the Akt signalling axis (Xiao et al. 2004; Xiao and Singh 2006a). In addition, Xiao et al provided experimental evidence to indicate that oral administration of DATS significantly retarded the growth of androgen-independent PC-3 xenografts in athymic mice without causing weight loss or any other side effects. The study also revealed that the DATS-mediated inhibition of PC-3 xenograft growth was correlated with the induction of multidomain proapoptotic proteins Bax and Bak (Xiao et al. 2006b). Hosono et al. (2005) also observed the antitumor activity of DATS in nude mice bearing HCT-15 xenografts.

Several individual compounds have been isolated from garlic. The main anticancer constituents of OSCs can be primarily divided into two groups. One group is lipid-soluble, such as diallyl sulfide (DAS), DADS and DATS, and the other group is water-soluble compounds [gamma]-glutamyl S-allylcysteine group such as S-allylcysteine (SAC) and S-allylmercaptocysteine (SAMC) (Herman-Antosiewicz and Singh 2004; Thomson and Ali 2003).

Garlic and related constituents appear to inhibit the initiation of carcinogenesis in multiple pathways, including the cell cycle arrest, the intrinsic pathway of cell apoptosis and anti-angiogenic pathway (Herman-Antosiewicz et al. 2007). Evidence is accumulating to indicate that garlic and related constituents can suppress proliferation of various cancer cells, but little is known about its inhibitory effect on human bladder cancer cells. In this study, we demonstrated that garlic derivatives, DATS, suppressed proliferation of T24 bladder cancer cells lines by inducing apoptosis through P13K/Akt pathway, thus down-regulating anti-apoptotic protein Bcl-2 and Bcl-xL, up-regulating pro-apoptotic protein Bax, leading to the activation of caspase-3 and poly(ADP-ribose) polymerase (PARP).

Materials and methods

Reagents and antibodies

DATS (98% purity) with commercial name as Allitridi, was purchased from Shanghai Hefeng Pharmacy Company (Shanghai, China). Tissue culture media and fetal bovine serum were from Gibco (Grand Island, NY). The annexin V-FITC apoptosis detection kit was from Beckman Coulter (Fullerton, CA). The antibodies against Akt, phosphorylated Akt (Ser473), PIP3-dependent kinase 1 (pPDKl) were purchased from Cell Signaling Technology (Beverly, MA). Primary antibodies to Bcl-2, Bax, Bcl-xL, caspase-3, [beta]-actin and poly (ADP-ribosyl) polymerase (PARP), and secondary antibodies were purchased from Santa-Cruz Biotechnology, Inc. (Santa Cruz, CA).

Cell culture and DATS treatment

The human bladder cancer cell line T24 was obtained from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences. The cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100mg/ml) in a humidified atmosphere with 5% [CO.sub.2] incubator at 37 [degrees]C. The culture mediums containing different concentrations of DATS were all freshly prepared at the time of each experiment. When cell confluence reached 50%-60%, the cells were treated with 10, 20, 40, or 80 [micro]M DATS, whereas the culture mediums treated cells served as control. After 24 h of treatment, photographs were taken using a phase-contrast microscope at 100 x magnification (Olympus, Japan).

Cell viability assay

The antiproliferative effect of DATS against different group cells was determined by using the MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] (Sigma, St. Louis, MO, USA) assay, as described previously (Qin et al. 2007). Briefly, T24 cells were seeded out in 96-well plates at a density of 1.0 x 104 cells per well. After overnight incubation, the cells were treated with DATS (0-80 [micro]M) for 24-72 h. At the various times following DATS treatment, the medium was removed and MTT (20 [micro]l of 5 mg/ml in PBS) was added to each well. After incubation for 4 h at 37 [degrees]C, the supernatant was removed and the formazan crystals solubilized by adding 150 [micro]l dimethyl sulfoxide. Viable cells were detected by measuring absorbance at 490 nm using MRX II absorbance reader (DYNEX Technologies, Chantilly, Virginia, USA). The reduction in viability of in DATS-treated T24 cells was expressed as a percentage compared to non-DATS treated control cells. Control cells were considered to be 100% viable. At least three independent experiments were performed.

Detection of apoptotic cells by flow cytometry

Cells were plated in 6-well plate (2 ml/well) at a density of 5 x [10.sup.5] cells/ml and incubated overnight. DATS with different concentrations were then added into each well and incubated for 24 h. The cells were collected and washed with PBS, followed by being resuspended in 1 x binding buffer at a concentration of 1 x [10.sup.6] cells/ml. One hundred microliters (1 x [10.sup.5] cells) of such solution was taken and mixed with 5 [micro]l of Annexin V-FITC and 5 [micro]l of Propidium Iodide (BD Biosciences, San Jose, CA, USA) according to the manufacturer's instruction. The mixed solution was incubated in the dark at room temperature for 15min, 400 [micro]l of 1 x dilution buffer were then added to each tube and cell apoptosis analysis was performed by Beckman Coulter FC500 Flow Cytometry System (Beckman Coulter, Fullerton, CA, USA) within 1 h.

Analysis of cell cycle distribution

Cell cycle analysis was performed using the Coulter DNA PrepTM Reagents Kit (Beckman Coulter, Fullerton, CA). Cells were prepared as mentioned above. The cells were then exposed to different concentrations of DATS for 24 h at 37 [degrees]C. Both floating and adherent cells were harvested, washed with cold PBS, fixed with 70% ethanol, and stored at 4 [degrees]C for subsequent cell cycle analysis. For detecting DNA content, cells were incubated in the dark at room temperature with 0.5 ml RNase A for 20 min and with 1 ml propidium iodide for 20 min. The DNA content of the cells was measured by a Beckman Coulter FC500 Flow Cytometry System. The percentage of cells in Gl, S and G2/M phases were calculated.

Western blot analysis

T24 cells treated with different concentrations of DATS for 24 h were harvested, rinsed twice with cold PBS, and lysed with lysis buffer. Following the centrifugation at 12 000g for 30 min at 4[degrees]C, the amount of protein in the supernatant was determined using the bicinchoninic acid protein assay. Equal amounts of protein (20-30 [micro]g) from cell lysate were subjected to 10%-15% poly-acrylamide gels and transferred to nitrocellulose membranes. Membranes were blocked with 5% defatted milk, and then incubated overnight with the appropriate primary antibody at dilutions specified by the manufacturer, followed by incubation at room temperature for 1 h with the corresponding horseradish peroxidase conjugated secondary antibody at 1:1000 dilution in TBST. Bound secondary antibody was detected using an enhanced chemiluminescence (ECL) system (Pierce Biotechnology Inc., Rockford, IL, USA).

Statistical analysis

All values are expressed as means [+ or -] SD. Statistical significance was compared between various treatment groups and controls using the one-way analysis of variance (ANOVA). A statistically significant difference was considered at the level of P<0.05.

Results

DATS inhibited T24 cell growth

In order to investigate the potential effects of DATS on growth and viability of human T24 cells, cells were treated with DATS at various concentrations and times by MTT assay. As shown in Fig. 1, inhibition of T24 cell proliferation by DATS was in a dose- and time-dependent manner. The [IC.sub.50] dose of DATS for 24 h for proliferation of T24 cells was approximately 31 [micro]M, whereas the [IC.sub.50] dose of DATS for 36 and 72 h was approximately 20 and 13 [micro]M, respectively.

[FIGURE 1 OMITTED]

DATS induced G2/M Phase cell cycle arrest in T24 cells

Based on the growth inhibitory response of DATS treatment in T24 cells, we next examined its effect on cell cycle distribution. T24 cells were treated with various concentrations of DATS (0-40 [micro]M) for 24 h and analyzed by flow cytometry. As shown in Fig. 2, the level of G2/M phase arrest was observed in a dose-dependent pattern. Treated with 0, 10, 20 and 40 [micro]M DATS for 24 h, the rate of G2/M phase cells were increased by 11.49%, 10.55%, 25.15%, and 43.53%, respectively. These results suggested that DATS can induce G2/M phase cell cycle arrest in T24 cells.

[FIGURE 2 OMITTED]

DATS induced apoptosis in T24 cells

To determine whether the DATS-induced growth inhibition was mediated by apoptosis, we further used the flow cytometry to identify the cell death types. Annexin-V-FITC/Pl plots profile is typically used to distinguish living cells (Annexin-V-FITC negative and PI negative), early apoptosis cells (Annexin-V-FITC positive and PI negative) and late apoptotic cells (Annexin-V-FITC positive and PI positive). As shown in Fig. 3A, T24 cells showed a dose-dependent apoptosis, including early as well as late apoptotic cell death. Approximately 6.2% of cells treated with 10 [micro]M DATS for 24 h underwent apoptosis, as well as 13.5% of cells treated with 20 [micro]M DATS, 16.6% of cells treated with 40 [micro]M DATS, and 20.0% of cells treated with 80 [micro]M DATS.

[FIGURE 3 OMITTED]

It is well known that caspases play important roles in the terminal execution of apoptosis induced by various stimulations (Salvesen and Dixit 1999). We then examined whether caspase-3 was activated during DATS-induced apoptosis. As shown in Fig. 3B, a dose-dependent decrease of pro-caspase-3 expression level was observed in DATS-treated T24 cells. PARP, the substrate of caspase-3, was regarded as another hallmark of apoptosis (Scovassi and Poirier 1999). As shown in Fig. 3B, the alteration of cleaved PARP level was consistent with that of caspase-3 and the 89 kDa cleaved PARP fragment was detected in DATS-treated samples.

The relationship between inhibition of Akt and the DATS-induced apoptosis in T24 cells

Akt is known as a kinase to promote cell survival and block apoptosis (Hemmings 1997; Kennedy et al. 1999). To determine whether the Akt activity is associated with the apoptotic effects of DATS, we examined the protein expression and phosphorylation level of Akt after DATS treatment. As given in Fig. 4, the levels of [Ser.sup.473], phosphorylated Akt and phospho-PDK1 was reduced in a dose-dependent manner. However, DATS treatment did not alter the level of total Akt protein expression. These results indicated that DATS-mediated inactivation of Akt could be involved in the anti-proliferative/pro-apoptotic response of T24 cells to DATS.

[FIGURE 4 OMITTED]

DATS treatment altered Bcl-2 family proteins

In order to get a deep insight into the mechanism of DATS-mediated apoptosis in T24 cells, we detected the protein level of Bcl-2 family proteins including Bcl-2 and Bax. The Western blot analysis data showed that the expression of Bcl-2 was significantly decreased in a dose-dependent manner after treatment with DATS. In contrast, the expression of Bax was significantly increased in a dose-dependent fashion (Fig. 5A). The ratio of Bax/Bcl-2 was significantly increased in a dose-dependent manner with DATS treatment (Fig. 5B).

[FIGURE 5 OMITTED]

Discussion

Bladder cancer currently remains one of the leading causes of cancer death throughout the world, and the 5-year survival rate in patients with invasive and metastatic bladder cancer remains very low. For these reasons, clinical development of agents that are nontoxic to normal cells but can treat, or delay onset and/or progression of human bladder cancer could have significant effects on disease-related cost and reducing the morbidity and mortality. Because many chemotherapeutic drugs have the ability to induce apoptosis in malignant cells, apoptosis has currently been a target for developing antitumor drugs (Kaufmann and Earnshaw 2000). In addition, there is a growing interest in natural herbal agents for the treatment or prevention of cancer because of their few side effects (Thatte et al. 2000).

Garlic was one of the plants with constituents having antitumor property and was used for treatment of tumors by Egyptians over 1550 years ago (Block 1985). Experimental and epidemiological studies provided evidence in support of association between garlic intake and reduced cancer risk (Khanum et al. 2004). In this study, our results demonstrated that DATS, a kind of garlic-derived compounds, decreased the T24 human bladder cancer cell number in a dose- and time-dependent manner which is related to induced G2/M phase cell cycle arrest and apoptosis.

The PI3K-Akt signaling pathway is believed to play an important role in the genesis of some human cancers (Vivanco and Sawyers 2002). Activation of the PI3K/Akt pathway confers chemotherapeutic resistance in numerous tumor types including bladder cancer (Fresno Vara et al. 2004). It has been found that 3-Phosphatidylinositol-dependent protein kinase (PDK)-l and integrin link kinase (ILK) can activate Akt by phosphorylating Thr308 and [Ser.sup.473], respectively (Xiao and Singh 2006a). Thereby inhibition of PI3K/Akt pathway is considered as a promising strategy for cancer treatment (Maddika et al. 2007). Recent studies have indicated that DATS inactivates Akt to trigger apoptosis in human prostate cancer cells and in human umbilical vein endothelial cells (Xiao and Singh 2006a; Xiao et al. 2006c). The present study demonstrated that DATS treatment caused a significant dose-dependent inhibition of elevated levels of phosphorylated PDK1 and a decrease in activating phosphorylations of Akt in T24 cells. Therefore our results provide the first evidence, to our knowledge, that Akt is constitutively active in T24 cells and that DATS can be a potential chemopreventive agent against bladder cancer by down-regulating Akt activity.

Akt regulates the process of cell survival by phosphorylating different substrates that directly or indirectly regulate the apoptotic program, such as inhibition of the pro-apoptotic Bcl-2 family member (Maddika et al. 2007). The majority of garlic-derived compounds activate the so called intrinsic (mitochondria-mediated pathway) in the execution of apoptosis. Activation of the intrinsic apoptotic pathway is regulated by the Bcl-2 family proteins (Herman-Antosiewicz et al. 2007). The Bcl-2 family proteins play critical roles in regulation of cell death processes (Adams and Cory 1998). Anti-apoptotic Bcl-2 family members (Bcl-2, BcI-XL) can block these mitochondrial events, whereas pro-apoptotic Bcl-2 family members (Bax, Bak, Bad) can trigger these changes. Our data show treatment of T24 cells with DATS down-regulates Bcl-2 proteins and up-regulates Bax proteins.

The caspases can be regarded as the central executors of the apoptotic pathway. Among the family of caspases, caspase-3 has been reported to be the most frequently activated caspase protease in apoptotic cells, indicating its crucial role in the cell death process (Cohen 1997). Caspase-3 activation causes the cleavage or degradation of several important substrates, including PARP. In this study, DATS effectively activated the expression of caspase 3, cleaved PARP and induced apoptosis in DATS treated T24 cells.

In summary, our in vitro studies suggested that DATS could inhibit on the growth of human T24 bladder cancer cells in a dose-and time- dependent manner. In addition, DATS was shown to induce apoptosis and G2/M arrest in bladder cancer cells. This apoptotic response was associated with the up-regulation of Bax, down-regulation of Bcl-2 and caspase-3 activation . Furthermore, inhibition of Akt pathways was involved in DATS-induced apoptosis. Taken together, DATS may be a promising chemopreventive and chemotherapeutic agent against bladder cancer, and further efforts are necessary to explore the in vivo effects of DATS.

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Yun-Bin Wang (a), (b), Jie Qin (a), Xiang-Yi Zheng (a), Yu Bai (a), Kai Yang (a), Li-Ping Xie (a), *

(a) Department of Urology. First Affiliated Hospital, School of Medicine, Zhejiang University, Qingchun Road 79, Hangzhou 310003, Zhejiang Province, China

(b) Department of Urology, First Affiliated Hospital, Inner Mongolia Medical College, Tongdaobei Road 1, Hohhot 010050, Inner Mongolia Autonomous Region, China

* Corresponding author. Tel.: +86 5718723 6735; fax: +86 5718707 2577.

E-mail address: xielp@zjuem.zju.eclu.cn (L.-P. Xie).

doi: 10.1016/j.phymed.2009.07.019
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Author:Wang, Yun-Bin; Qin, Jie; Zheng, Xiang-Yi; Bai, Yu; Yang, Kai; Xie, Li-Ping
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9CHIN
Date:Apr 1, 2010
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