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Anti-Cancer Drugs Effects of Gambogic Acid on Regulation of Steroid Receptor Coactivator-3 in Lung Adenocarcinoma A549 Cells Rui Li*, Yan Chen**, Wen-xiu Shu, Fei Zhao, Yuan Liu, Lu Wen Department of Hematology, Union Hospital, Tongji Medical College, Huazhong University Of Science and Technology, Wuhan 430022, China

CLC number: R73-36 Document code: A Article ID: 1000-9604(2009)01-0068-06 DOI: 10.1007/s11670-009-0068-x

ABSTRACT

Objective: To investigate the effects of gambogic acid (GA) on the proliferation and apoptosis of Human lung adenocarcinoma A549 cells in vitro, as well as the regulation of steroid receptor coactivator-3 (SRC-3) to explore the relationship between them.

Methods: The effect of GA on the growth of A549 cells was studied by MTT assay. Apoptosis was detected through Hoechst 33258 staining. RT-PCR and Western blot technologies were applied to assess the expression of SRC-3, whereas, the localization of SRC-3 was determined by using confocal microscopy method.

Results: GA presented striking proliferation inhibition potency on A549 cells in vitro in a time- and dose-dependent manner, with the IC50 value for 24 h was 3.17±0.13 µmol/L. Hoechst 33258 staining showed that GA could induce apoptosis in A549 cells. Over-expression of SRC-3 was found in A549 cells, whereas the mRNA and protein expression levels of SRC-3 were significantly downregulated in A549 cells induced by GA in a dose-dependent manner. The disposition of SRC-3 was situated mainly at the nuclear. Conclusion: GA may exert its strong anti-leukemia effects through the regulation of the expression of SRC-3. It may be a new target for the therapy of lung cancer. Key words: Gambogic acid; Lung cancer; Apoptosis; SRC-3

INTRODUCTION Gamboge is a dry resin secreted from Garcinia

hanburryi, and gambogic acid (GA, C38H44O8, molecular weight: 628.75) is the main active compound of gamboge[1, 2]. Recently, in vitro and in vivo studies showed that GA had strong anti-tumor effect on several tumors. However, the molecular mechanisms of its anti-tumor activity and its effects on human lung adenocarcinoma A549 cells are still poorly understood and await further investigations. Steroid receptor coactivator-3 (SRC-3) is a member of the p160 family of nuclear receptor coactivators, which also 111111 ⎯⎯⎯⎯⎯⎯⎯⎯⎯ Received: Oct. 10, 2008; Accepted: Jan. 5, 2009 This work was supported by the National Natural Science Foundation of China (No. 30472267). *E-mail: lirui-514@163.com **Corresponding author. E-mail: yanchen@public.wh.hb.cn

includes SRC-1 and SRC-2. A growing body of evidence has revealed that overexpression of SRC-3 might promote initiation and/or progression of carcinogenesis[3]. Mechanisms that regulate SRC-3 may provide novel opportunities for drug development in lung cancer treatment. In this study, we investigated the anti-tumor effects of GA and its relation to the regulation of SRC-3 in A549 cells.

MATERIALS AND METHODS Drugs and Reagents

GA (C38H44O8, molecular weight 628.75), was purchased from the Calbiochem (SanDiego, USA), and was initially dissolved in dimethylsulfoxide (DMSO), stored at -20°C, and then thawed before use. MTT, DMSO, PI and

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Hoechst 33258 were purchased from Sigma. DMEM medium and fetal bovine serum (FBS) were purchased from Gibco (USA). Anti-SRC-3 antibody was purchased from Cell signaling Technology Inc. (Beverly, Massachusetts, USA). Antibodies specific to β-actin and HRP-conjugated secondary antibodies were all from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Chemiluminescence (ECL) reagent kit was purchased from Pierce Biotechnology (Rockford, IL, USA). Trizol reagent was from Invitrogen Company, USA. RT-PCR kit was purchased from Fermentas, Lithuania, USA. A549 cell line was obtained from the China Center for Typical Culture Collection (Wuhan, China) and maintained in DMEM medium supplemented with 10% fetal bovine serum at 37°C in a humidified atmosphere. 3-[4, 5-Dimethylthiazol-2-yl]-2, 5-diphenyl- tetrazolium Bromide (MTT) Assay

The antiproliferative effect of GA against A549 cells was determined by MTT method. Briefly, the A549 cells were plated at a density of 104 cells per well in 96-well plate and allowed to attach overnight. The cells were incubated with various concentrations of GA for 0, 24, 48, 72 h in triplicate. Thereafter, 20 µl MTT solution (5 mg/ml) was added to each well. After continued incubation for 4 h, the supernatant was discarded and 150 µl of DMSO was added. When the blue crystals were dissolved, the optical density (OD) at 492 nm was read with a 96-well multiscanner autoreader (Biotech Instruments, NY, USA). The following formula was used: cell proliferation inhibited (%) = [1–(OD of the experimental samples/OD of the control)] ×100%. Hoechst 33258 Staining

Nuclear fragmentation was visualized by Hoechst 33258 staining of apoptotic nuclei. A549 cell suspension was partitioned into the wells of 6-well plates with sterile glass cover slips and cultured. When the cells had grown to monolayer, 3.0 µmol/L (close to IC50) of GA was added to the wells. After 24 h cultures, the apoptotic cells were washed, and then fixed in 4% paraformaldehyde for 10min at room temperature. The cells were permeabilized with 0.1% Triton X-100 for 5 min at room temperature, and then incubated with Hoechst 33258 for 30 min at 37°C, rinsed with PBS and mounted on slides with glycerol-PBS. Finally, the cells were viewed with an Olympus BH-2 fluorescence microscope (Tokyo, Japan).

Immunofluorescence with Confocal Microscopy

For the immunofluorescence experiments, cells were fixed in 4% paraformaldehyde for 10 min and permeabilized with 0.2% Triton X-100 on ice for 10 min. The samples were blocked with 3% bovine serum albumin plus 0.02% Triton X-100 in PBS for 30 min, incubated with antibody against SRC-3 (at a working dilution of 1:50) separately overnight at 4°C, then, FITC-labeled secondary antibody diluted in PBS was applied for 60 min. PI (50 µg/ml) was used to visualize the DNA for 15 min. Coverslips were mounted with polylysine. Images were captured using a FV500 confocal microscope (Olympus, Tokyo, Japan).

RNA isolation and Reverse Transcription-PCR

Total RNA was prepared. cDNA was synthesized according to the manufacture’s instruction of Fermentas kit. 25 µl PCR reaction mixture was amplified at 94°C for 5 min, which was followed by 10 cycles at 94°C for 30 s and at 60°C for 1 min, then by 30 cycles at 94°C for 30 s, 53°C for 40 s, 72°C for 1 min, with a final extension at 72°C for 7 min. The following primer pairs were designed from human cDNA sequences available in GenBank and synthesized by Shanghai Invitrogen Biotechnology Co., Ltd, China: SRC-3 (GenBank accession no. NM_181659): 5-TGTTT- CCGTCTCGATTCACCA-3 and 5-GATTAGGAGA- AAAC TTGGATCC-3; β-actin (GenBank accession no. NM_001101), 5-CTGTCCCTGTATGCCTCTG- 3 and 5-ATGTCACGCACGATTTCC-3. After amplification, 5 µl aliquots of products were resolved on a 1.5% agarose gel. DNA bands were quantified by Smart View Bio-electrophoresis Image Analysis System. The ratio between the target gene and β-actin gene band densities was used for quantitative evaluation. Preparation of Cell Lysates and Western Blot Analysis

After treatment, A549 cells were harvested and lysed in 100 µl of lysis buffer by incubation on ice for 30 min, then, the extracts were centrifuged at 12,000 r/min for 15 min at 4°C. After addition of sample loading buffer, protein samples were electrophoresed on a 8% SDS-polyacrylamide gel and then transferred onto nitrocellulose membranes. The membranes were blocked in non-fat milk for 60 min at room temperature, washed, and incubated with anti- SRC-3 antibody (dilution 1:1000) and anti-β-actin (dilution 1:400). After overnight

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incubation at 4°C, the blots were washed, and exposed to HRP-conjugated secondary antibodies (dilution 1:3000) for 1h. Finally, the blots were washed and detected with ECL substrate solution. Densitometric analysis was performed using Quantity One software. Statistical Analysis

All the experiments were repeated at least three times. All data were expressed as x±s, and t-test was used for statistical analysis by SPSS 13.0. P values <0.05 was considered as significant different.

RESULTS Effects of GA on the Proliferation of A549 Cells

The cytotoxicity of GA to A549 cells was calculated from the loss of cell viability using MTT assay. As shown in Figure 1, treatment with various concentrations of 0, 0.25, 0.5, 1.0, 2.0, 4.0, 8.0 µmol/L GA for 0, 24, 48, 72 h resulted in a significant decrease in cell viability in a dose- and time-dependent manner. The IC50 values for 24 h and 48 h were 3.17±0.13 µmol/L and 1.85±0.08 µmol/L, respectively. With the exposure time increased, the IC50 values decreased gradually.

Figure 1. Antiproliferative effect of GA on A549

cells by MTT assay

Effects of GA on Apoptosis of A549 Cells

To address the role of GA in conferring sensitivity to apoptosis, A549 cells were exposed to GA (3.0 µmol/L) for 24 h, and then detected with Hoechst 33258 staining. As shown in Hoechst

33258 staining figures (Figure 2), GA induced apoptotic cell death in A549 cells. Apoptotic bodies containing nuclear fragments were found in GA-treated cells, and the chromatin becomes condensed and marginalized, the nuclear envelope appeared lytic and the cytoplasm became shrinkaged, while the normal A549 cells presented intact plasma membrane and order chromatin folding.

Figure 2. Induction of apoptosis by GA in A549 cells by Hoechst 33258 staining assay

A: negative control, untreated by GA; B: cells treated by 3.0 µmol/L GA for 24 h, the

arrows represented the apoptotic nuclear fragmentations. Disposition of SRC-3 in A549 Cells

To visualize the subcellular locations of SRC-3 better, confocal microscope was used. We found that SRC-3 appeared high fluorescence intensity in nucleus, with an average optical density (OD) value being 180.02±7.16. However, the fluorescence density dropped to 85.86±4.96 (P<0.05) when cultured with 3.0 µmol/L GA (Figure 3).

Effects of GA on the Regulation of SRC-3 Protein Expression in A549 Cells

To study the mechanisms of anti-tumor activity by GA, we first examined whether there existed constitutive deregulation of SRC-3 protein in A549 cells. We found that high protein level of SRC-3 appeared in A549 cells compared with GA-untreated cells, whereas the protein expression of SRC-3 was down-regulated dramatically in a dose-dependent manner in GA-treated cells (Figure 4). Effects of GA on the Regulation of SRC-3 mRNA Expression in A549 Cells

In view of the decrease in SRC-3 protein level,

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we then tried to find whether the decreased protein level was mediated at the transcriptional level by a decrease of SRC-3 mRNA in GA-treated cells. As we presumed, high mRNA expression of SRC-3 was found in control groups, with a mean value of 0.92±0.04. After treatment with GA, the mRNA expression of SRC-3 in A549 cells declined in a dose-dependent manner, with the mean values of 0.82±0.02, 0.58±0.06, and 0.31±0.04, respectively significantly decreased in comparison with the control (P<0.05, Figure 5).

Figure 3. Effects of GA on the subcellular

localization of SRC-3 in A549 cells Cells were treated with 3.0 µmol/L GA for 24 h, and

PI (50 mg/L) was used to visualize the DNA (which appeared as red). The expression and subcellular location of SRC-3 in control or GA –treated groups were visualize via a FV500 confocal microscope (which appeared as green) (× 400).

A and B: control group, C and D: 3.0 μmol/L GA-treated group.

Figure 4. Effects of GA on the mRNA expression of

SRC-3 in A549 cells Lane 0: marker; Lane 1: negative control untreated

by GA; Lane 2, Lane 3 and Lane 4: cells treated by 1.5 µmol/L, 3.0 µmol/L and 4.5 µmol/L GA for 24 h, respectively.

DISCUSSION

GA, a major active compound extracted from the gamboge resin of Garcinia hanburryi, was selected for further study due to its potent antitumor activities. It has been well documented that GA has been observed to inhibit tumor cell growth or induce cell apoptosis in several mammalian cell culture systems. However, the effects and mechanism of action of GA on human lung adenocarcinoma A549 cells are not well known. In this study, we demonstrated that GA could inhibit the proliferation and induce apoptosis in A549 cells in vitro, and explored the possible relation between its anti-tumor effects and the regulation of SRC-3.

Figure 5. Effects of GA on the protein expression of

SRC-3 in A549 cells Lane 1: negative control untreated by GA; Lane 2,

Lane 3 and Lane 4: cells treated by 1.5 μmol/L, 3.0 μmol/L and 4.5 µmol/L GA for 24 h, respectively.

In the present study, we showed that GA

exerted significant inhibition of the proliferation of A549 cells in a time-dependent and dose-dependent manners, the inhibition rate reached 93.1±0.68% after exposure to GA 8 µmol/L for 72 h. A median inhibitory concentration (IC50) for a contact time of 24 h was 3.17±0.13 µmol/L. Moreover, GA also presented potent apoptosis-induction activity with typical morphological changes associated with apoptosis including nucleus disruption and apoptotic body formation. In other tumor cell lines, such as hepatocellular carcinoma, gastric cancer, leukemia, GA also had strong apoptosis-inducing activity. It was suggested that the underlying anti-tumor mechanisms of GA might be associated with the inhibition of telomerase activity, or with the change in the expression of some pivotal genes, such as Bax and Bcl-2[4, 5]. In our previous study, we also found that GA induced leukemia T cell apoptosis by downregulation of NF-kappaB, and activation of caspase proteins[6].

Steroid receptor coactivator-3 (SRC-3, also known as AIB1, NCoA3, pCIP, RAC3, ACTR and

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TRAM1), localized in a frequently amplified chromosomal region, 20q12[7], was first identified as Amplified in Breast cancer 1 (AIB1). Extensive investigations reveal that SRC-3 interacts with nuclear receptors[8, 9] and certain other transcription factors (NF-κB and AP-1)[10, 11], recruits histone acetyltransferase (CBP and p300) and methyltransferases (CARM1 and PRMT1) for chromatin remodeling and facilitates target gene transcription[12, 13]. Since its important roles in cell growth, reproduction, metabolism, and cytokine signaling, any changes of concentrations and activities in SRC-3 may greatly affect the expression levels of many genes related to SRC-3, and as a consequence, influence a variety of cellular processes. More recently, increasing evidence has revealed that SRC-3 gene is highly overexpressed in several tumors, including breast cancer, ovarian cancer, gastric cancer, pancreatic cancer and hepatocellular carcinoma[14−19]. Moreover, progressive increased frequency of SRC-3 expression was detected during pancreas cancer progression[16]. Furthermore, SRC-3 was also closely associated with metastasis and tumor recurrence in gastric cancer and hepatocellular carcinoma[18, 19]. Transgenic mice overexpressing SRC-3 were found to have an extremely high tumor incidence[20]. In contrast, SRC-3 knockout mice had a significantly lower incidence of mammary gland tumor genesis[14]. All these results indicate that SRC-3 has a central role in tumor genesis and progression.

Additional researches revealed that SRC-3 also played an important role in oncogenic processes of human non-small cell lung cancer[21]. Lahusen et al. reported that SRC-3 was overexpressed in human non-small cell lung cancer cell line A549 and down-regulation of SRC-3 resulted in decreased tumor cell proliferation[22]. Thus, SRC-3 might become a new target for anti-tumor drug in treatment of lung cancer. To get some insight into the anti-tumor mechanism of GA, we performed experiments to determine whether GA had impact on the expression of SRC-3. In this study, we found SRC-3 was overexpressed in A549 cells, as previously proved by Lahusen. However, in GA treated cells, the mRNA and protein levels of SRC-3 dropped in a dose-dependent manner, suggesting that GA induced the down-regulation of SRC-3 in A549 cells, thereby lead to apoptosis of A549 cells. We also found SRC-3 was situated extensively in the nucleus. However, the fluorescence density was significantly reduced when treated with GA.

In summary, current data strongly suggested

that GA had potent effects on growth-arrest and apoptosis-induction in A549 cells in vitro, which might be related to the downregulation of SRC-3. It suggests that GA may become a new drug to treat lung cancer.

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