Inhibition of cell-cycle progression in human colorectal carcinoma Lovo cells by andrographolide

Chemico-Biological Interactions 174 (2008) 201–210

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Inhibition of cell-cycle progression in human colorectal carcinoma Lovo cells by andrographolide Ming-Der Shi b,c , Hui-Hsuan Lin b , Yi-Che Lee a , Jian-Kang Chao d , Rong-An Lin e , Jing-Hsien Chen a,∗ a

Graduate Institute of Biological Science and Technology, College of Medicine and Life Science, Chung Hwa University of Medical Technology, No. 89, Wen Hwa 1st Street, Rende Shiang, Tainan County 717, Taiwan b Department of Medical Technology, College of Medicine and Life Science, Chung Hwa University of Medical Technology, Tainan, Taiwan c Pathology and Laboratory Medicine, Yongkang Veterans Hospital, Tainan, Taiwan d Department of Psychiatry, Yongkang Veterans Hospital, Tainan, Taiwan e Department of Pharmacy, Yongkang Veterans Hospital, Tainan, Taiwan

a r t i c l e

i n f o

Article history: Received 17 January 2008 Received in revised form 4 June 2008 Accepted 8 June 2008 Available online 20 June 2008 Keywords: Andrographolide Cell-cycle arrest Human colorectal carcinoma Cyclin Cdk Rb

a b s t r a c t In recent years, attention has been focused on the anti-cancer properties of pure components, an important role in the prevention of disease. Andrographolide (Andro), the major constituent of Andrographis paniculata (Burm. F.) Nees plant, is implicated towards its pharmacological activity. To investigate the mechanism basis for the anti-tumor properties of Andro, Andro was used to examine its effect on cell-cycle progression in human colorectal carcinoma Lovo cells. The data from cell growth experiment showed that Andro exhibited the anti-proliferation effect on Lovo cells in a time- and dose-dependent manner. This event was accompanied the arrest of the cells at the G1–S phase by Andro at the tested concentrations of 0–30 ␮M. Cellular uptake of Andro and Andro was confirmed by capillary electrophoresis analysis and the intracellular accumulation of Andro (0.61 ± 0.07 ␮M/mg protein) was observed when treatment of Lovo cells with Andro for 12 h. In addition, an accumulation of the cells in G1 phase (15% increase for 10 ␮M of Andro) was observed as well as by the association with a marked decrease in the protein expression of Cyclin A, Cyclin D1, Cdk2 and Cdk4. Andro also inducted the content of Cdk inhibitor p21 and p16, and the phosphorylation of p53. Further immunoprecipitation studies found that, in response to the treatment, the formation of Cyclin D1/Cdk4 and Cyclin A/Cdk2 complexes had declined, preventing the phosphorylation of Rb and the subsequent dissociation of Rb/E2F complex. These results suggested Andro can inhibit Lovo cell growth by G1–S phase arrest, and was exerted by inducing the expression of p53, p21 and p16 that, in turn, repressed the activity of Cyclin D1/Cdk4 and/or Cyclin A/Cdk2, as well as Rb phosphorylation. © 2008 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Abbreviations: Andro, andrographolide; Lovo, human colorectal carcinoma; Cdk, cyclin-dependent kinase; CKI, Cdk inhibitor; Rb, retinoblastoma; DMSO, dimethyl sulfoxide; TBS, tris-buffered saline; FACS, fluorescence-activated cell sorting; RNase, ribonuclease; SDSPAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; ECL, enhanced chemiluminescence; GI50 , the molar concentration that produces 50% growth inhibition; ROS, reactive oxygen species. ∗ Corresponding author. Tel.: +886 6 2674567x402; fax: +886 6 2902371. E-mail address: [email protected] (J.-H. Chen). 0009-2797/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2008.06.006

Andrographis paniculata (Burm. F.) Nees (Acathaceae) is an important herbal medicine widely used in China, India and other Southeastern Asian countries. The main components of A. paniculata are the diterpene lactones of which andrographolide (Andro; Fig. 1A) is the major component and constitutes 70% of the plant extract fraction [1]. Andro has been reported to have multiple pharmacological properties, including anti-inflammatory [2,3], anti-allergic

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[4], anti-platelet aggregation [5], hepatoprotective [6,7] and anti-HIV activities [8,9]. And this compound has been widely used in clinic for the treatment of fever, cold, inflammation, diarrhea and other infectious diseases. Recent studies suggested that Andro is an interesting pharmacophore with anti-cancer and immunomodulatory activities and hence has the potential to be developed as a chemotherapeutic agent [10,11]. Andro can be readily isolated in high yield and has anti-tumor effects against breast cancer models [10], whose anti-cancer activity is thought to be exerted through blockage of cell-cycle progression by the induction of Cyclin-dependent kinase inhibitors (CKIs) and with a concomitant decrease in Cyclin-dependent kinase (Cdk) expression [10,12]. Multiple genetic changes taking place during the process of carcinogenesis cause the abnormalities of cells. Recent advances in cell biology have illustrated the detailed mechanisms of the cell-cycle regulatory systems and have shown that an up-regulated cellular proliferation is a common characteristic in numerous cancers [13,14]. Eukaryotic cells have developed precise and well-regulated mechanisms to control progression through the cell cycle [15]. Regulation of the vertebrate cell cycle requires the periodic formation, activation, and inactivation of unique protein kinase complexes that consist of Cyclin (regulatory) and Cdk (catalytic) subunits. The associations of Cyclin D1 and Cdk4, Cyclin E, and Cdk2, and Cyclin A and Cdk2

have also been shown to phosphorylate retinoblastoma (Rb) in the G0–G1 and the G1–S phase transitions of the cell cycle [16]. Upon phosphorylation, Rb releases and activates a number of proteins such as the E2F family of transcription factors at the G1–S transition phase [17,18], which in turn regulates the expression of several genes involved in DNA replication, such as dihydrofolate reductase, thymidine kinase, and DNA polymerase [19]. Regulation of G1 Cyclin/Cdk activity is also dependent on CKIs, which can bind and inactivate Cyclin/Cdk complexes [20,21]. Several inhibitory proteins have been identified, including p27, p16, and p21, which have been reported to mediate G1 cell-cycle arrest [22]. Another major factor believed to plays a central role in cell-cycle regulation is p53. The major downstream effectors of p53 include p21 and Cyclin D1, which participate in cell-cycle arrest. The diverse phosphorylation sites of p53 have been demonstrated to play important roles in the regulation of many cellular responses, of which the phosphorylation of p53 at serine 15 is an important target for p53 activation and stabilization. Recent reports have revealed that some compounds could reactivate the p53 function to inhibit cancer cell proliferation through cell-cycle arrest and/or apoptosis, which opens new possibilities to fight cancer [23]. Cancer cells differ from normal mortal cells in that they are no longer regulated by the usual growth controlling mechanisms. Most of the anti-cancer agents currently in use are inducers of apoptosis, necrosis, cell-cycle arrest and cell differentiation; others might involve immunostimulating activity. Many traditional herbs have been reported to have these activities, but it appears likely that different pathways are involved in different types of cells [24,25] and at different concentrations. Andro, an herbal medicine exhibiting anti-inflammatory properties, was found to suppress breast cancer cell proliferation [10]. In the present study, we report evidences demonstrating that Andro inhibited human colorectal carcinoma Lovo cell-cycle progression by G1–S arrest. 2. Materials and method 2.1. Chemicals

Fig. 1. (A) Chemical structure of Andro. (B) Effects of Andro on Lovo cell growth. Cultured cells were treated with various concentrations (0, 5, 10, 20 and 30 ␮M) of Andro for 6, 12, 24, and 48 h as described in the text. The number of cells was counted by trypan blue dye exclusion assay. The results were represented the mean ± S.D. of three independent experiments and the significant difference was established at p < 0.05. *p < 0.05, **p < 0.005 compared with the control group for the indicated time. DMSO served as the solvent control.

Andrographolide (purity 98%), Tris–HCl, EDTA, SDS, phenylmethylsulfonyl fluoride, bovine serum albumin (BSA), leupeptin, nonidet p-40, deoxycholic acid, sodium orthovanadate, and aprotinin were purchased from the Sigma–Aldrich Chemical Co. (St. Louis, MO). Protein assay kits were obtained from Bio-Rad Labs. (Hercules, CA). F12 nutrient mixture, fetal-calf serum, trypsin-EDTA, and penicillin, streptomycin, and neomysin mixture (PSN) were purchased from Gibco/BRL (Gaithersburg, MD). Rb, E2F-1 monoclonal antibodies were purchased from BDPharMingen (San Diego, CA). Cyclin A, Cyclin D1, Cyclin D3, Cdk2, Cdk4, p16, p21, p27 and p53 monoclonal and Cyclin E polyclonal antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The phosphoSer15 p53 and phospho-Ser795 Rb rabbit polyclonal antibodies were purchased from Cell Signaling technology (Beverly, MA). Horseradish peroxidase-conjugated

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anti-rabbit and anti-mouse secondary antibodies were purchased from NEN Life Science Products, Inc. (Boston, MA). 2.2. Cell culture Human colorectal carcinoma Lovo cells were maintained as monolayers in F12 nutrient mixture supplemented with 10% heat-inactivated fetal-calf serum and PSN (100 units/ml penicillin and 10 ␮g/ml streptomycin) at 37 ◦ C in a humidified atmosphere of 95% air/5% CO2 . 2.3. Preparation of stock solution A stock solution of Andro in dimethyl sulfoxide (DMSO) at a concentration of 100 mM was prepared and protected from light and stored at −20 ◦ C. Before use, Andro solution was freshly prepared by diluting with medium to the desired concentrations. Control experiments received the same volume of solvent DMSO (final concentration of 0.2%). 2.4. Cell growth experiment Cells (1 × 105 cells/ml) were seeded in 12-well plates and treated with Andro at various concentrations (5–30 ␮M) or DMSO as control for 6, 12, 24, and 48 h. The final concentration of DMSO in all the cultures was 0.1%. The number of cells was measured by the trypan blue dye exclusion assay. 2.5. Capillary electrophoresis analysis

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2.7. Preparation of cell extract and immunoblot analysis To prepare whole-cell extract, cells were washed with PBS containing zinc ion (1 mM), and then suspended in a lysis buffer (50 mM Tris, 5 mM EDTA, 150 ␮M sodium chloride, 1% Nonidet P-40, 0.5% deoxycholic acid, 1 mM sodium orthovanadate, 81 ␮g/ml aprotinin, 170 ␮g/ml leupeptin, and 100 ␮g/ml phenylsulfonyl fluoride; pH7.5). After 30 min of mixing at 4 ◦ C, the mixture was centrifuged at 10,000 × g for 10 min, and the supernatant was collected as whole-cell extract. Protein content of the samples was determined with the Bio-Rad protein assay reagent using BSA as a standard. For Western-blotting analysis, whole-cell extracts (30 ␮g protein) from control and Andro-treated samples were resolved on 10% SDS-PAGE gels along with pre-stained protein molecular weight standards (Bio-Rad). The separated proteins were then blotted onto NC membrane (0.45 ␮m, Bio-Rad) and reacted with primary antibodies (against Cyclin A, Cyclin D1, Cyclin D3, Cyclin E, Cdk2, Cdk4, p27, p21, p16, p53, phosphop53, Rb, phospho-Rb, E2F, and ␤-actin as internal control). After washing, the blots were incubated with peroxidaseconjugated goat anti-mouse antibody. Immunodetection was carried out using the ECL Western-blotting detection kit (Amersham Corp., U.K.). Relative protein expression levels were quantified by densitometric measurement of ECL reaction bands and normalized with values of ␤actin. 2.8. Immunoprecipitation Cell lysates were prepared using the above lysis buffer. 500 ␮g of protein from cell lysates was pre-cleared with protein A-Sepharose (Amersham Pharmacia Biotech), followed by immunoprecipitation using monoclonal antiCdk2, -Cdk4, and -E2F (Santa Cruz Biotech) antibodies. Immune complexes were harvested with protein A, and immunoprecipitated proteins were analyzed by SDS-PAGE, as above. Immunodetection was carried out using monoclonal anti-Cdk2, -Cdk4, -Rb, -E2F, -Cyclin A, -Cyclin D1 and -CyclinD3; polyclonal anti-Cyclin E antibodies.

Following incubation of Andro and Andro (10 ␮M) with Lovo cells for 6, 12 and 24 h, cells were washed twice with PBS and then the method for the determination of intracellular Andro [26,27]. Cell pellets were resuspended with 10 ␮l of 1% Triton X-100 and 10 ␮l of 1 N HCl, vortexed vigorously and allowed to stand at 4 ◦ C for 20 min. Samples were analyzed by Beckman Coulter P/ACETM MDQ Capillary Electrophoresis System, according to the following conditions. Mobile phase – 100 mM sodium borate buffer (pH 8.3); capillary – eCAPTM capillary tubing with total length – 60.2 cm, effective length – 50.2 cm, I.D. – 75 ␮m; O.D. – 375 ␮m; injection – 0.5 psi for 5 s; temperature – 25 ◦ C; voltage – 25 kV; detection – target signal at 214 nm.

Results were reported as means ± S.D., and statistical analysis was obtained using an unpaired t-test. A value of p < 0.05 was considered statistically significant.

2.6. Cell-cycle analysis

3. Result

Flow cytometric analysis of Lovo cells was performed using a FACScan (Becton Dickinson Immunocytometry Systems, UK). To analyze the cell-cycle distribution, cells were first treated with Andro, and then trypsinized and resuspended in 70% absolute ethanol. After an incubation at −20 ◦ C for at least 24 h, the cells were resuspended in 1 ml of cell-cycle assay buffer [0.38 mM sodium citrate, 0.5 mg/ml ribonuclease (RNase) A and 0.01 mg/ml propidium iodide] at dark room for 15 min. Cell-cycle analysis was carried out by a flow cytometer and analyzed with the ModFit LT 3.0 software (Verity Software, Topsham, ME).

3.1. Effects of Andro on Lovo cell viability

2.9. Statistical analysis

In preliminary experiments, the effect of Andro on the proliferation of Lovo colorectal carcinoma cells was evaluated. The proliferation assay was performed by MTT assay, and found that Andro showed much efficacy of anti-cell proliferation abilities. The GI50 (the molar concentration that produces 50% growth inhibition) value of Andro was 8.6 ␮M for 24 h incubation (data not show). In view of the potential anti-tumor abilities of the compound (0, 5, 10, 20 and 30 ␮M) for the indicated time (0, 6, 12, 24 and

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3.3. Andro-induced cell-cycle arrest and alterations in cell-cycle regulatory proteins in Lovo cells

Fig. 2. Capillary electrophoresis determination of Andro incorporated into Lovo cells. Data was presented from three independent experiments.

48 h), the cell viability was further evaluated by trypan blue dye exclusion assay, the result was shown in Fig. 1B. Higher concentrations of Andro (10–30 ␮M) almost completely inhibited the cell proliferation for 24–48 h. To evaluate the bioavailability of Andro, the cytosolic concentration of Andro (10 ␮M) was detected by capillary electrophoresis method. As shown in Fig. 2, the incorporation of the sample into Lovo cells was very quickly and reached the limitation within 12 h. The cytoplasmic concentration of the sample, expressed in ␮M/mg protein, ranged from 0.35 and 0.61 for 6 and 12 h to 0.53 for 24 h incubation. 3.2. Effects of Andro on Lovo cell-cycle distribution To further determine the possible involvement of Andro in the regulation of cell cycle, the effects of Andro (0, 5, 10, 20 and 30 ␮M) on colorectal carcinoma cells were analyzed by flow cytometry. As shown in Fig. 3A, a 24-h treatment of 10 ␮M Andro-caused an apparent accumulation of the cells in the G1 phase (from 43.28% to 56.05%), more than approximately 15% increase. Further, Lovo cells were treated with 10 ␮M Andro at the indicated time (0, 6, 12 and 24 h) and collected for the analysis of cell-cycle distribution. Andro increased the G1 fraction (from 42.55% to 54.05%) while decreasing the S fraction in the cells in a time-dependent manner (Fig. 3B). These results suggested that Andro inhibited cell-cycle progression by causing arrest at the G1–S phase.

In mammalian cells, Cyclins comprise an extensive family of proteins whose cell-cycle-dependent synthesis is postulated to control multiple events during the cell cycle [20,21]. To investigate further the mechanism of the effect Andro on cell-cycle arrest at G1 phase, Lovo cells treated with 10 ␮M Andro for the indicated times were subjected to immunoblotting analysis. We first analyzed the protein levels of Cyclin family, including Cyclin A, Cyclin D1, Cyclin D3, and Cyclin E. Among them, Cyclin A and Cyclin D1 levels were reduced by Andro treatment at 6 and 24 h, respectively (Fig. 4A). Cyclin D1 is one of the major regulators of the G1–S transition. A decrease of its expression thus suppresses entry into the S phase of cell cycle. On the contrary, there were no changes in the protein expressions of Cyclin D3 and Cyclin E. Furthermore, Cdks play a critical role in the commitment of a cell to proliferate [15]. The expression of Cdk family proteins (Cdk2 and Cdk4) was then examined. The protein levels of Cdk2 and Cdk4 were reduced by Andro treatment at 6 and 24 h, respectively (Fig. 4B). Cell-cycle transition from G1 to S requires the temporal activation of Cyclin D1/Cdk4, Cyclin E/Cdk2, and Cyclin A/Cdk2 [16]. To investigate how Andro-reduced Cyclins and Cdks expression and inhibitd cell-cycle progression, we analyzed the level of Cyclin A/Cdk2, Cyclin E/Cdk2 and Cyclin D1/Cdk4 complexes by immunoprecipitation. In agreement with the supposition, Andro treatment decreased the formation of Cyclin A/Cdk2 and Cyclin D1/Cdk4 complexes (Fig. 5) at 6 and 24 h. On the contrary, there was no change in Cyclin E/Cdk2 complex. Taken together, we concluded that the Androinduced cell-cycle arrest at the G1–S phases is associated with the Cyclin A/Cdk2 and/or Cyclin D1/Cdk4 proteins. 3.4. Effects of Andro on protein level of CKIs in Lovo cells Regulation of Cyclin/Cdk activity is also dependent on CKIs, which can bind to and inactivate Cyclin/Cdk complexes [20,21]. Several inhibitory proteins have been identified, including p27, p16, and p21, which have been reported to mediate G1 cell-cycle arrest [22]. To verify if Andro-reduced G1–S Cyclin-Cdk activity was dependent on the activation of CKIs, Lovo cells were treated with Andro (10 ␮M) for the indicated times. We found that expression of p21 and p16 were induced and reached the max levels by Andro treatment at about 3.75- and 2.14-folds (p < 0.005), as shown in Fig. 6A, and that there was no significant alternation in p27. Because Andro significantly increased the amount of p21, which is one of the p53 target genes [23], we next examined whether the Andro-induced cell-cycle arrest is mediated by the activation of p53. The cellular levels of phospho-p53 (Ser15) and p53 showed an increase about 2.09-folds (p < 0.005) and 1.37-folds (p < 0.05) when AGS cells were treated with 10 ␮M of Andro for 6 and 12 h, respectively (Fig. 6B). These results indicated that the Andro-induced cell-cycle arrest was associated with an

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Fig. 3. Effects of Andro on Lovo cell-cycle progression. (A) Cultured cells were treated with or without Andro 0, 5, 10, 20, 30 ␮M. Twenty-four hours later, cell cycle was analyzed by flow cytometry. (B) Cultured cells were treated with 10 ␮M Andro for the indicated time (0, 6, 12 and 24 h), washed and then harvested. The cells were fixed and stained with propidium iodide and the DNA content was analyzed by flow cytometry (FACS). The cell number percentage in each phase (sub-G1, G1, S, and G2/M) of the cell cycle was calculated and expressed. Data was presented from three independent experiments.

induction of p53 protein level and p53 phosphorylation that subsequently modulated the expression of p21.

for cell proliferation (Fig. 7B). These results indicated that Rb/E2F complex was involved in the Andro-induced cellcycle block at G1 phase.

3.5. Effects of Andro on Rb phosphorylation and E2F protein expression

4. Discussion

Cyclin D1/Cdk4 plays a major role in initiation of the cell cycle, passage through the restriction point (G0), and entry into the S phase. The only known target of active Cyclin D1/Cdk4 is Rb protein; however, other Cdks, such as Cyclin E/Cdk2, and Cyclin A/Cdk2 have also been shown to phosphorylate Rb in the G1 phase and the G1–S transition of the cell cycle [18]. Cyclin/Cdk complexes phosphorylate the Rb protein, releasing E2F from its sequestration by Rb and allowing E2F to transactivate genes essential for the S phase [17]. To elucidate the role of Rb in the Andro-induced Lovo cells arrest, we assessed the phosphorylated state of Rb and its complex with E2F. The results showed that the expression of Rb protein was only slightly increased, even though the level of phospho-Rb (Ser795) was decreased by 10 ␮M Andro treatment (Fig. 7A). A decrease in the phospho-Rb was correlated to an increase in Rb/E2F complex in cell cycle. Using immunoprecipitation, we confirmed that the addition of Andro up-regulated the formation of Rb/E2F complex in Lovo cells at 6–24 h. An increase in the Rb/E2F complex prevented the release of E2F transcription factor and, thus, prevented the transcription of the genes required

Andrographolide (Andro), a diterpenoid lactone isolated from a traditional herbal medicine A. paniculata, is known to possess multiple pharmacological activities. Previous studies reported that Andro in comparison to other two common diterpenoids of the herbal medicine, dehydroandrographolide and neoandrographolide, had more potent anti-cancer activity against human leukemia HL-60 cells and other cancer cells. It is hypothesized that the differences may be attributed to the chemical structure that influences their cytotoxic properties [28]. In contrast with our and the previous findings, it was found Andro was more growth inhibitory in HL-60 cells with GI50 near to 6.9 ␮M, next to Lovo cells (GI50 = 8.6 ␮M, Fig. 1B), than the other cancer cell lines, including MCF-7 and HepG2, at a 24-h of treatment [28–30]. These studies cooperatively demonstrated that Andro revealed the strongest potency to induce cytotoxicity toward cancer cells. The finding that some analogs of Andro could be totally inactive has important implications because there are recent concerns regarding the stability of diterpene lactones. Andro has been reported to gradually convert

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Fig. 4. Time course of Andro treatment on (A) Cyclins and (B) Cdks expression in Lovo cells. Cultured cells were treated with Andro (10 ␮M) for the indicated times. The Cyclins and Cdks proteins were analyzed by Western blotting. The quantitative data were presented as means ± S.D. of three repeats from one independent experiment. *p < 0.05; **p < 0.005, compared with control at the respective times.

to 14-deoxy-11,12-didehydroandrographolide during storage, and this process is accelerated by heat [31]. In our study, to eliminate the possibility that Andro was naturally metabolized to other diterpenoids during the experimental process, the bioavailability of Andro was evaluated and the cytosolic concentrations of Andro was detected by capillary electrophoresis method. The data showed that the maximum incorporation of Andro into Lovo cells was reached a maximum after 12 h of incubation (Fig. 2). Although there are some studies of the bioavailability of Andro in human and animal models [27,32], this is the first study reported that the uptake of Andro in human colorectal cell. Recently, Andro has been found to be able to inhibit cancer cell proliferation [29], induce cell-cycle arrest [10,29] and promote apoptosis [28,33] in human cancer cells. For example, Andro treatment induced mitochondrial cytochrome c release, accompanied by increased expression level of Bax and decreased expression level of Bcl-2 protein in HL-60 cells [28]. In PC-3 cells, Andro-caused

apoptotic morphological change and caspase 3, 8 activations [33]. However, the molecular mechanism underlying Andro-induced cell-cycle arrest in cancer cells has not been fully investigated. Look back on previous studies, Andro was found to cause G0/G1 cell-cycle arrest through induction of p27 and decreased expression of Cdk4 in MCF-7 and HL60 cells [28,29]. Recently, Li et al. have reported that Andro have an anti-cancer activity brought about by reducing the level and activity of Cdc2, thus indicating a G2/M arrest. And the study concluded that the cytotoxic effect of Andro on HepG2 is primary attributed to the induction of cell-cycle arrest and a late apoptosis via an alteration of cellular redox status [30]. Another synthesis of Andro derivative (14acetylandrographolide) showed non-specific phase of the cell-cycle arrest in MCF-7 cells treated, which might involve targeting phosphatases or kinases that regulates cell cycle [12]. To date, the molecular mechanism of cell-cycle arrest by Andro seemed to involve single active protein and lack a causal link between these proteins, but the cell-signal cascades remain obscure. Hence, further studies to clarify

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Fig. 5. Analysis of Cyclin/Cdk complexes in Lovo cells following Andro treatment: cell extracts prepared from Lovo cells at the times indicated following treatment with Andro (10 ␮M) were immunoprecipitated with Cdk2 and Cdk4. The precipitated complexes were examined for immunoblotting using Cyclin A, Cyclin E, and Cyclin D1 antibodies. The quantitative data were presented as means ± S.D. of three repeats from one independent study; *p < 0.05; **p < 0.005, compared with control at the respective times.

Fig. 6. Expression of cell-cycle inhibitors in Lovo cells following Andro treatment. Cell lysates were prepared from Lovo cells at the times indicated following treatment with Andro (1 ␮M). (A) The expression of p27, p21, p16; (B) phospho-p53 and p53 were analyzed by Western blotting. The quantitative data were presented as means ± S.D. of three repeats from one independent study; *p < 0.05; **p < 0.005, compared with control at the respective times.

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Fig. 7. Time courses of Andro treatment on Rb expression and phosphorylation (Ser 795) in Lovo cells. Cultured cells were treated with Andro (10 ␮M) for the indicated times. (A) The levels of phospho-Rb (Ser795) and E2F were analyzed by Western blotting. (B) Analysis of Rb/E2F complex in Lovo cells following Andro treatment at the time indicated. Cell extracts (500 ␮g) were immunoprecipitated with E2F. The precipitated complexes were examined for Rb by immunoblotting. The quantitative data were presented as means ± S.D. of three repeats from one independent experiment. *p < 0.05; **p < 0.005, compared with control at the respective times.

the whole pathway, including kinases and checkpoints of cell-cycle phase are needs. To this goal, we examined the detail mechanisms of Andro-induced cell-cycle arrest by focusing on the protein/protein interaction (such as Cyclin/Cdk and Rb/E2F) and the regulatory role of the CKI family members. As shown in Figs. 4–6, Andro-induced the expression of p21, p16 and p53 and decreased the Cyclin A, Cyclin D1, Cdk4 and Cdk2 protein levels compared with control at the respective time. Among these changed proteins, p21 was the most susceptible to Andro (p21 expression was increased to about 3.75-fold of control under the same treatment condition; Fig. 6A). The increased expression of p21 by Andro is of significance because decreased p21 expression has been associated with aggressive phenotype in many cancers. It has already been demonstrated that ectopic expression of p21 results in cell-cycle arrest and apoptosis in cancer cells dependent of p53 status [23]. p21 also mediates the cell-cycle arrest and apoptosis caused by different anticancer agents [34–36]. For example, curcumin increases the expression of p21 as well as its down-regulating Cyclin D1 and Cyclin E leading to G1 arrest in cell-cycle progression in human prostate and breast cancer cells [34,35]. Epigallocatechin gallate (EGCG) has also been shown to induce G1 arrest in melanoma cell lines via induction of p21 and by down-modulation of Cdk2 kinase activity [36]. The grape seed extract up-regulates p21 and down-regulates Cdk2, CDK4, and Cyclin E leading to G1 arrest in human prostate carcinoma cells [37]. In this study, we observed similar results with Andro in Lovo cells with wild-type p53. Many phytochemicals like cruciferous glucosinolate indole-3-carbinol, tea polyphenol EGCG, and soy isoflavone

genistein have been shown to inhibit the growth and proliferation of cancer cells (with intact p53) by increased expression of p53, resulting in cell-cycle arrest [38]. p53 activates p21, which is a key regulator of G1 arrest and also contributes to G2–M arrest in cell cycle by inhibiting Cdc2 or by its binding to Cyclin B [39]. The cell-cycle arrest and induction of apoptosis by dependent of p53 is significant because wild-type/activation of p53, although happens in half of the human cancers allow cancer cells to circumvent the intrinsic and extrinsic controls that tightly regulate the cell cycle, cell division, and apoptosis and also confers sensitive to chemotherapy and radiotherapy [40]. On the other hand, Li et al. observed that Andro-induced cell-cycle arrest at the G2/M phase and late cell death in HepG2 cells via an intrinsic pathway to decrease the level of GSH and by means of excessive accumulation of intracellular reactive oxygen species (ROS), mainly H2 O2 [30]. The Andro-caused the over-produced of H2 O2 was an upstream event of the loss of mitochondrial membrane potential and the expression of p53. It is believed that ROS have divergent effects according to the cell types and it triggers redox signaling pathways, including oxidative stress, loss of cell function, cell-cycle arrest and apoptosis [41]. However, Andro was identified to be an antioxidant role responsible for the enhancement of cellular antioxidant defense in the previous studies [42,43]. Whether the characteristics of induced cell-cycle arrest or apoptosis observed in HepG2 (cancer cells) are also applied to the protective function of Andro on rat primary hepatocytes (normal cells) remains unknown. Cancer cells differ from normal mortal cells in that they are no longer regulated by the usual growth controlling mechanisms. Most of the anti-

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cancer agents currently in use are inducers of apoptosis, necrosis, cell-cycle arrest and cell differentiation; others might involve immunostimulating activity. Many traditional herbs have been reported to have these activities, but it appears likely that different pathways are involved in different types of cells [24,25] and at different concentrations. As our study showed that in Lovo cells, Andro inhibited cell-cycle progression (Fig. 3) and induced p53 expression and phosphorylation (Fig. 6B), which might be triggered by the alteration of ROS. Our findings, therefore, are in agreement to some proven evidences that extrusion of GSH alters the intracellular redox state. However, further investigations have to be conducted regarding the association of the G1 arrest with phospho-p53 and an alteration of ROS in response to Andro treatment. The results from this study suggested that Andro can inhibit Lovo cell growth by G1–S phase arrest, and was exerted by inducing the expression of p53, p21 and p16 that, in turn, repressed the activity of Cyclin A/Cdk2 and/or Cyclin D1/Cdk4, as well as Rb phosphorylation and the subsequent release of E2F, which failed to target genes that trigger cells to progress from G1 to S phase (Fig. 8). More importantly, p53 is a critical mediator relaying the p21 might be binding and inactivating the Cdk2, 4 leading to their degradation with a concomitant decrease in the levels

Fig. 8. A proposed model for the Andro-mediated cell-cycle dysregulation of human colorectal carcinoma Lovo cells.

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of Cyclin A and E. To our knowledge, this is the first evidence demonstrating the chemopreventive agent Andro-induced a CKI–Cyclin–Cdk network and cell-cycle arrest program. To understand the mechanism better we are currently trying to determine the compound molecular target of Andro that leads to a block in the G1–S phase of the cell cycle, bringing about the anti-cancer activity. Further studies on definitive mechanisms of the cancer chemotherapeutic activities of Andro in animal models, and even clinical trials are needed.

Acknowledgement This work was supported by the grant from Yongkang Veterans Hospital, Tainan, Taiwan.

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