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Carcinogenesis Advance Access originally published online on September 28, 2006
Carcinogenesis 2007 28(3):632-638; doi:10.1093/carcin/bgl168
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Gambogic acid-induced G2/M phase cell-cycle arrest via disturbing CDK7-mediated phosphorylation of CDC2/p34 in human gastric carcinoma BGC-823 cells

Jun Yu{dagger}, Qing-Long Guo{dagger}, Qi-Dong You1,*, Li Zhao, Hong-Yan Gu, Yong Yang, Hai-wei Zhang, Zi Tan2 and Xiaotang Wang2,*

Department of Physiology, China Pharmaceutical University Nanjing 210009, People's Republic of China
1 Department of Medicinal Chemistry, China Pharmaceutical University Nanjing 210009, People's Republic of China
2 Department of Chemistry and Biochemistry, Florida International University Miami, FL 33199, USA

*To whom correspondence should be addressed. Q.Y., Tel: +86 25 83271351; Fax: +86 25 83271055; Email: anticancer_drug{at}yahoo.com.cn;X.W., Tel: +1 305 348 7544; Fax: +1 305 348 3772; Email: wangx{at}fiu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Molecular mechanisms of cell-cycle arrest caused by gambogic acid (GA), a natural product isolated from the gamboge resin of Garcinia hanburryi tree, have been investigated using BGC-823 human gastric carcinoma cells as a model. Based on our 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazoliumbromide (MTT) assay and flow cytometric analysis, treatment of BGC-823 cells with growth suppressive concentrations of GA caused an irreversible arrest in the G2/M phase of the cell cycle. Western blot analysis demonstrated that GA-induced cell-cycle arrest in BGC-823 cells was associated with a significant decrease in CDC2/p34 synthesis, which led to the accumulation of phosphorylated-Tyr15 (inactive) form of CDC2/p34. Real-time PCR, western blot and kinase activity assays revealed that GA-induced reduction of CDC2/p34 expression was mediated through the inhibition of cyclin-dependent kinase (CDK)-activating kinase (CDK7/cyclin H) activity. In addition, GA-treated cells were shown to have a low level of CDK7 kinase-phosphorylated-Thr161 CDC2/p34 (active). Taken together, our results suggested that the inhibited proliferation of GA-treated BGC-823 cells was associated with the decreased production of CDK7 mRNA and protein, which in turn, resulted in the reduction of CDK7 kinase activity. The reduced CDK7 kinase activity is responsible for the inactivation of CDC2/p34 kinase and the irreversible G2/M phase cell-cycle arrest of human gastric carcinoma BGC-823 cells.

Abbreviations: GA, gambogic acid; CAK, CDK-activating kinase; CDK, cyclin-dependent kinase; MPF, mitosis-promoting factor; PBS, physiologically buffered saline


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Gambogic acid (GA) is the major active ingredient of gamboge, a brownish to orange resin exuded from Garcinia hanburryi tree in Southeast Asia (1,2). The structure of this natural product (Figure 1) has been firmly established from both detailed NMR spectroscopic analysis (2) and X-ray crystallographic studies (3), however the molecular mechanism of its potent anticancer activity remains poorly understood and warrants further investigations (4–7).


Figure 1
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  		Fig. 1 Molecular structure and atom numbering scheme of GA (C38H44O8, Mol. Wt.: 628.75).

 
We have previously demonstrated that the potent anticancer activity (both in vitro and in vivo) of GA is mainly attributed to its activation of the impaired apoptotic pathways in cancerous cells via down regulation of telomerase activity (4,8,9). More recently, we have reported that GA exhibited significant inhibitory effect on cultured human gastric carcinoma cell lines including BGC-823, MGC-803 and SGC-7901 (9,10). Although cell-cycle disruption had been suggested to be the possible mechanism for GA's inhibitory effect on these cell lines, details of the mechanisms of cell-cycle arrest caused by GA are not fully understood.

CDC2/p34 is a cell-cycle kinase responsible for the regulation of G2 progression and G2/M transition in all eukaryotic cells (11–13). The active mitotic kinase or mitosis-promoting factor (MPF) is a heterodimer comprised of a catalytic subunit CDC2/p34 and a positive regulatory subunit, a B-type cyclin. However, the activity of the CDC2/p34 kinase depends not only on its association with cyclin B, but also on its phosphorylation state. Phosphorylation of either Thr14 or Tyr15 inhibits CDC2/p34 kinase activity (14,15). Previous studies have also confirmed that CDK7/cyclin H complex or CDK-activating kinase (CAK) is able to regulate the activity of a number of different CDKs through phosphorylation of a conserved threonine residue at the active site of these kinases.

The present study was undertaken to gain insights into the mechanisms through which GA inhibits cell-cycle progression using BGC-823 human gastric carcinoma cells as a model. We demonstrate that GA-induced cell-cycle arrest was associated with the accumulation of phosphorylated-Tyr15 (inactive) form of CDC2/p34 that was caused by the inhibited CDK7 kinase activity in GA-treated human gastric cancer cells.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Materials
Gambogic acid (GA) was isolated and purified according to the established methods with slight modifications (9). GA with 99% purity was dissolved in dimethyl sulfoxide (DMSO) and was used in all experiments. RPMI-1640 medium was purchased from Gibco, USA. Heat-inactivated fetal bovine serum (FBS) was provided by Sijiqing Company Ltd, China. Penicillin and streptomycin were supplied by Lukang Pharmaceutical Company Ltd, China. MTT were obtained from Sigma, USA. TriPure isolation reagent and DNase-free RNase were purchased from Roche, USA. RT–PCR kit was obtained from TaKaRa, Japan. Fluorophore SybrGreen I was obtained from Roche Diagnostics, USA. All antibodies (anti-CyclinB1, -CDC2/p34, -Cyclin H and -CDK7 monoclonal antibodies; anti p-CDC2/p34 (Tyr15) and p-CDC2/p34 (Thr161) polyclonal antibodies) were purchased from Santa Cruz Biotech Ltd, USA.

Cell culture
Human gastric cell line BGC-823 was purchased from Cell Bank of Shanghai Institute of Biochemistry and Cell Biology. The cells were grown in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml benzylpenicillin and 100 µg/ml streptomycin in a humidified environment with 5% CO2 at 37°C in the presence of GA at desired concentrations or 0.1% DMSO alone for control purposes.

MTT assay of growth inhibition
BGC-823 cells were cultured in RPMI 1640 media as described above till mid-log phase. Cells were harvested by centrifugation at 250x g for 5 min and re-suspended in RPMI-1640 + 10% FBS to make a stock cell suspension containing 2 x 105 cells/ml. To the wells of a 96-well plate, 100 µl of this stock cell suspension was then added. GA was weighed and diluted with DMSO to make a 2 mM stock solution. This stock solution was further diluted with culture media to make a series of secondary stock solutions so that addition of 1 µl of the secondary solutions will result in seven different final concentrations (0.50, 1.0, 2.0, 3.0, 4.0, 5.0 and 6.0 µM) of GA. Triplicate experiments were performed in a parallel manner for each concentration point and the results were presented as mean ± SD. Controls were performed in which only culture media and DMSO were added. Media was then added to bring the total volume of each well to 200 µl. The cells were then incubated at 37°C in a 5% CO2, 95% air atmosphere. After 6, 12, 24 and 48 h of incubation, the culture medium was removed and the cells washed twice with physiologically buffered saline (PBS). Then 20 µl of 5 mg/ml MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide] was added to each well. The cells were further incubated at 37°C for 4 h. The supernatant was discarded and 100 µl of DMSO was added to each well. The mixture was shaken on a micro-vibrator for 5 min and the absorbance was measured at 570 nm that served as a measure of cell viability. Inhibition ratio (I%) was calculated using the following equation:

Formula

The IC50 was taken as the concentration that caused 50% inhibition of cell proliferation.

Cell-cycle analysis
Logarithmic cells were dispersed with 0.02% EDTA to prepare a 1 x 106 cells/ml cell suspension. Cells treated with DMSO or different concentrations of GA for different period were trypsinized and washed once with PBS, and fixed in 100% ethanol for 1 h at –20°C. Fixed cells were washed with PBS before incubation with 0.5 ml PBS containing 0.05% RNase and 0.5% Triton X-100 for 30 min at 37°C. The cells were then stained with propidium iodide (PI; Gibco) before DNA content was determined. DNA content and cell cycle were determined using a FACScan laser flow cytometer (FACSCalibur, Becton Dickinson, USA). The data were analyzed using the software MODFIT and CELLQUEST.

Western blot assay
At the end of GA treatment attached cells were washed twice with PBS and cell lysates were prepared in non-denaturing lysis buffer as described previously (5). For immunoblot analysis, equal amounts of total cellular protein (30 µg) were denatured in 2x SDS–PAGE sample buffer and subjected to electrophoresis on 12% Tris–glycine gel. The separated proteins were transferred onto nitrocellulose membranes followed by blocking with 5% non-fat milk powder (w/v) in tris-buffered saline Tween-20 (TBST, 10 mM Tris, 100 mM NaCl, and 0.1% Tween-20) for 1 h at room temperature or over night at 4°C. The membranes were then incubated with one of the following antibodies: anti-CDC2/p34 monoclonal, anti-cyclin B1 monoclonal, anti-cyclin H or anti-CDK7 monoclonal antibodies (Santa Cruz Biotechnology), anti p-CDC2/p34 (Tyr15) and p-CDC2/p34 (Thr161) polyclonal antibodies; followed by peroxidase-conjugated appropriate secondary antibody, and visualized by the ECL detection system (Amersham).

Immunoprecipitation and in vitro histone H1 kinase assay
Cell lysates were prepared for immunoprecipitation. Aliquots of cell lysate were incubated with anti-CDK7 monoclonal antibody and anti p-CDC2/p34 (Thr161) polyclonal antibody at 4°C for 15 h. The immune complexes were precipitated by addition of protein-A-Sepharose (Sigma Chemical) The immunoprecipitates were then reacted with 5 µg histone H1 (Calbiochem) in kinase buffer consisting of 20 mM Tris–HCl (pH 7.4), 7.5 mM MgCl2, 1 mM dithiotheitol (DTT), 10 mM ATP, 10 mCi [{gamma}-32P]ATP (Amersham). Kinase reaction was performed by shaking at room temperature for 30 min after separation by 10% SDS–PAGE. The gels were subsequently dried and autoradiographed.

RT–PCR and Quantitative PCR assay
Total cellular RNA was extracted from GA (at IC50) treated BGC-823 cells using the TriPure Solution following the manufacture's instructions. The purity of the RNA extracted was determined by the ratio of A260/A280 using a BioPhotometer (Eppendorf, Germany). Reverse transcription–polymerase chain reaction (RT–PCR) was performed following the protocol supplied with TaKaRa kit. The amplified PCR products were separated by electrophoresis on a 2% agarose gel containing ethidium bromide and quantitated by relative intensities of the bands as compared to those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using Gel Base/Gel Blot/Gel Excel/Gel Sequence analysis software (UVP, UK). A value of 100% was given to the relative intensity of untreated cells (control). Primers for CDK7 were designed against the portion of human CDK7 mRNA (Genbank accession no: NM_001799 [GenBank] ) with the assistance of the Primer Premier 5 software. The sequences of the PCR primers and the expected size of amplicons were as follows: 5'-ATTCGTGTTGTCCTGGGAGC-3' (sense), 5'-GGCCTTGTAAACGGTGGC-3' (anti-sense), 145 bp.

Quantitative PCR was performed using the above primers according to the protocol supplied by the manufacturer (ABI PRISM 7500 Realtime System; Applied Biosystems). Quantification of the amplified product was done on a cycle-by-cycle basis via the acquisition of a fluorescent signal generated by binding of the fluorophore SybrGreen I (Roche Diagnostics) to double-stranded DNA. The cycle number at which the fluorescence signal crosses a certain threshold (threshold cycle (CT) in correlation with the background fluorescence of the assay) was noted. This CT value is proportional to the logarithm of the target DNA concentration in the assay. From a dilution series of a DNA amount corresponding to a known concentration of cells, a standard curve was produced in which the CT was plotted versus the logarithm of the starting concentration of DNA corresponding to a known number of cells, with each cell containing multiple copies of the targeted ITS region. The volume of the PCR mixture was 10 µl comprising 1x Fast Start Taq DNA polymerase mixture (Roche Diagnostics), 3 mM MgCl2, 0.5 µM primers, and 1 µl DNA extract. The same concentrations of MgCl2 and primers were found to be optimal for the primer pair. The standards and the samples were run in duplicate on the ABI PRISM7500 instrument. Amplification of the product was visualized in the quantification curve analysis. The specificity of the amplified products was confirmed by a melting curve analysis.

Statistical analysis
All data were expressed as mean ± SD and statistically compared by one-way ANOVA with Dunnett's test or unpaired Student's t-test in different experiments, P < 0.05 was taken as statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Effects of GA on the inhibition of cell proliferation
Exponentially growing BGC-823 cells were cultured continuously in the absence or presence of different concentrations of GA. The effects of GA on cell growth were assessed by the commonly used MTT assay at varying intervals (6, 12, 24, 48 and 72 h) of treatment. As shown in Figure 2, GA treatment significantly inhibited the growth of BGC-823 cells. The degree of growth inhibition depends on both the concentration and the length of treatment. For example, at a given duration of treatment, the number of viable cells decreases as the concentration of GA increases. On the other hand, when GA concentration is held constant, the number of viable cells decreases regularly as the exposure time increases. The effect of GA treatment is statistically significant as compared with the control group (P < 0.01, unpaired t-test). A linear regression of the data in Figure 2 allowed the prediction of the IC50 (2.30 ± 0.26, 1.41 ± 0.15 and 1.02 ± 0.05 µM for 24, 48 and 72 h treatment, respectively) of GA for BGC-823 cells.


Figure 2
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  		Fig. 2 Inhibitory effect of GA on the proliferation of BGC-823 cells. The results shown were the mean of three parallel experiments (triplicate wells) for each concentration point.

 
GA treatment caused G2/M phase cell-cycle arrest in BGC-823 cells
To test whether GA could affect the cell cycle of BGC-823 cells, asynchonized cells treated with DMSO or GA (1.4 µM, IC50) for 6, 12, 24 and 48 h were subjected to flow cytometric analysis after DNA staining. Representative histograms for cell-cycle distribution in BGC-823 cells following exposure different concentrations of GA are shown in Figure 3. The effects of GA on BGC-823 cell-cycle distribution are summarized in Table I. It can be seen that a 24-h exposure of BGC-823 cells to growth suppressive concentrations of GA (1.4 and 2.0 µM) resulted in a statistically significant increase in G2/M fraction that was accompanied by a decrease in G0/G1 cells. For example, the percentage of G2/M fraction was increased by ~1.7- and 2.1-fold on treatment of BGC-823 cells with 1.4 and 2.0 µM GA, respectively, when compared with DMSO-treated control (Table 1). In time course experiments using 1.4 µM (IC50) GA, the G2/M phase cell-cycle arrest was evident as early as 12 h after treatment, and persisted for the duration of the experiment (Figure 3). These results indicated that the inhibitory effect of GA against proliferation of BGC-823 cells correlated with G2/M phase cell-cycle arrest.


Figure 3
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  		Fig. 3 Representative histograms depicting cell-cycle distribution in BGC-823 cell cultures treated with DMSO (control) or various concentrations of GA for different period of time. (A) 0.1% DMSO (control), (B) 1.4 mM GA for 12, 24 and 48 h, (C) different concentration (1.0, 1.4 and 2 µm) for 48 h, and (D) semi-quantification of the diploid analysis using a FACScan laser flow cytometer. All data presented were representatives of at least three independent experiments, P < 0.05.

 


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  		Table I Effects of 24 h GA treatment on BGC-823 cell-cycle distribution as compared with DMSO

 
Effects of GA treatment on the expression of cell-cycle-regulatory proteins
Eukaryotic cell-cycle progression involves sequential activation of CDKs, whose activity is dependent upon their association with regulatory cyclins (16). A complex between CDK1 and cyclin B1 is important for entry into mitosis in most organisms (16,17). The activity of CDK1/cyclin B1 kinase is negatively regulated by reversible phosphorylations at Thr14 and Tyr15 of CDK1 (16). Dephosphorylation of Thr14 and Tyr15 of CDK1, and hence activation of CDK1/cyclin B1 kinase complex, is believed to be a rate-limiting step for entry into mitosis (18). We determined the effect of GA treatment on levels of CDK7, cyclins (B1, H, D, E and A), DNA damage checkpoint markers (Chk1/2 and Cdc25), and CDC2 proteins by immunoblotting to gain insights into the mechanism of cell-cycle arrest in our model. As can be seen in Figure 4, treatment of BGC-823 cells with GA did not increase the protein level of cyclinB1 at 12–48 h time points. The level of CDK7 protein was decreased in GA-treated BGC-823 cells after 24 and 48 h treatment. Exposure of BGC-823 cells to GA also resulted in a decrease in the protein level of CDC2-phospho (Thr161). The GA-mediated decrease in CDC2-phospho (Thr161) protein level was observed as early as 24 h after treatment, and persisted for the duration of the experiment. These results suggested that the GA-induced cell-cycle arrest in BGC-823 cells was caused, at least in part, by changes in the CDC2-phospho (Thr161), CDK7 and cyclin H. No significant changes were observed for other cell-cycle marker proteins, such as cyclin D, E and A, and DNA damage checkpoint markers (Chk1/2 and Cdc25).


Figure 4
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  		Fig. 4 Expression of cell-cycle-regulatory proteins in GA-treated cells. The left panel denotes the effect of 0.01% DMSO (control) and the right panel represents the results of 1.4 µM GA after exposure for 12, 24 and 48 h. At the end of each treatment, total cell lysates were prepared, and an equal amount of protein was subjected to SDS–PAGE. Western blot analyses were done with anti-CDC2/p34-phospho(Thr161), CDC2/p34-phospho(Tyr15), -cyclin H, -CDK7, -cyclin B1 and ß-actin primary antibodies as described in Meterials and methods. Data shown were representatives of at least three independent experiments.

 
Effects of GA on CDK7 kinase activities and CDC2/p34 phosphatase activities
Since CDC2/p34 kinase is the key regulator that promotes mitosis, we further investigated the underlying mechanism for the inhibition of CDC2/p34 kinase activity in BGC-823 cells treated with GA and examined the possible involvement of key CDC2/p34 regulator, CDK7 kinase. CDK7 kinase was immunoprecipitated from the extracts of BGC-823 cells exposed to 1.0, 1.4 and 2.0 µM GA. These CDK7 kinase preparations were each mixed with CDC2/p34 immunoprecipitates from untreated exponentially growing BGC-823 cells. Following incubation, CDC2/p34 kinase activity was examined using an in vitro kinase assay. As shown in Figure 5, the CDC2/p34 kinase activity of the CDK7 kinase preparations from GA-treated cells decreased systematically as the concentration of GA was increased. Furthermore, exposure to 1.4 µM GA for 12 h prevented the phosphorylation of CDC2/p34 at Thr161. Even at 48 h Thr161-phosphorylated CDC2/p34 was still at the basal level (Figure 5B). These findings indicated that the inhibitory effect of GA on CDC2/p34 kinase activity was mediated though the inhibition of CDK7 kinase. Thus, we concluded that GA treatment prevented the exit of BGC-823 cells from the G2/M phase by disruption of CDK7 kinase activity and inactivation of CDC2/p34 kinase.


Figure 5
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  		Fig. 5 Effect of 1.4 µM GA on CDK7 kinase (upper lane) and CDC2/p34 phosphatase (lower lane) activity. CDK7 kinase activity was determined by histone H1 kinase assay as described in Materials and methods section. The duration of treatment is given at the bottom of the gel. Data shown were representatives of at least three independent experiments.

 
Effect of GA treatment on the levels of CDK7 mRNA
Based on the observed decrease in CDK7 protein levels, we next evaluated whether this decrease was mediated at the transcription level by a decrease in CDK7 mRNA in GA treated BGC-823 cells. As shown in Figure 6, CDK7 mRNA level decreased significantly in cells treated with 1.4 µM GA compared with that in cells from the control group (at 24 h P < 0.05, and at 48 h P < 0.01, one-way ANOVA with Dunnett's test). Similar results were observed when cells were treated with different concentrations of GA (1.0, 1.4 and 2.0 µM) for a fixed period of exposure time.


Figure 6
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  		Fig. 6 Down regulation of CDK7 mRNA in GA-treated BGC-823 cells. Cells were treated with either DMSO or GA (1.4 µM) for 12, 24 and 48 h. (A) Gel of RT–PCR product from cells treated with 1.4 µM GA for 12, 24 and 48 h. The rightmost lane (labeled as c) denotes control experiment. (B) RT–PCR analysis for CDK7 mRNA synthesis performed as described in Materials and methods section. (C) Quantification of the RT–PCR from three independent experiments, P < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
We have shown previously that GA treatment caused apoptosis in several human cancer cell lines including human gastric cancer cell lines BGC-823 and MGC-803 (5,8,9). Our results suggested that GA-induced apoptosis in these cell lines were associated with changes in the expression level of some pivotal proteins, such as Bax and Bcl-2. These results prompted us to further examine whether there are any differentially expressed genes that contribute to the GA-induced apoptosis or cell-cycle arrest.

Cell-cycle progression in eukaryotic cells is strictly regulated by a class of cyclins and cyclin-dependent kinases (CDKs). Cyclins are positive regulatory subunits for CDKs. The complex formation of cyclins with CDKs results in an active agent that phosphorylates substrates involved in cell-cycle progression. The cyclin B, in association with CDC2/p34, governs cell-cycle progression through enhanced cell-cycle distribution in G2/M fraction. The activity of cyclinB/CDC2/p34 complexes is subjected to positive and negative regulation. Both cyclin binding and phosphorylation by CAK are required for the activation of this complex. CDK7 can phosphorylate CDKs that promote cell-cycle progression, and has been shown to serve as a CAK. Phosphorylation of either Thr14 or Tyr15 inhibits CDC2/p34 kinase activity, while phosphorylation of Thr161by CDK7 kinase is required for kinase activity.

This study revealed that GA-induced G2/M arrest (Figure 3) and the inactivation of CDC2/p34 was achieved through inhibition of CDK7 (Figure 4) in gastric carcinoma BGC-823 cells. Data presented herein indicated that GA-treated human gastric carcinoma BGC-823 cells were arrested irreversibly in G2/M phase of the cell cycle, which was accompanied by changes of the expression and activity of cell-cycle-regulatory proteins: a marked decrease in the level of CDC2/p34, CDK7 and cyclin H (Figure 4). Western blot using anti-phospho-CDC2/p34 antibody revealed a significant increase in the level of p-Tyr15 CDC2/p34 and marked decrease in the level of p-Thr161 CDC2/p34 in GA-treated cells.

Furthermore, GA treatment prevented the dephosphorylation of Tyr15 of the CDC2/p34 kinase or phosphorylation of Thr161 of the CDC2/p34 kinase which resulted in kinase inactivation. The hyperphosphorylation of the kinases responsible for phosphorylation of CDC2/p34 at Thr14/Tyr15 is one of the known mechanisms for G2 arrest, the dephosphorylation of the kinases responsible for phosphorylation of CDC2/p34 at Thr161 is the other known mechanism for G2 arrest. All these suggested that GA treatment affects the activity of CDC2/p34/cyclin B1 kinase by causing accumulation of phosphorylated Thr14/Tyr15 CDC2/p34 (inactive) and dephosphorylation of Thr161 CDC2/p34 (active). This implication is supported by a decline in the level of CDK7 mRNA and CDK7/cyclin H protein. In addition, kinase activity assay and real-time PCR indicated a significant increase in the level of CDK7 kinase and CDK7 mRNA in GA-treated cells. These results indicated that GA could block the cell cycle in the G2 phase and offered an explanation for the inhibited proliferation of BGC-823 cells. The activity of CDC2/p34 is dependent upon CDK7 kinase activities. The results reported in this work suggested that GA was an effective repressor of cyclin H and CDK7, resulting in decreased CDK7 activity. This explains why GA could inhibit the proliferation of many different types of human carcinoma cells.

In conclusion, we demonstrated that the inhibited proliferation of GA treated BGC-823 cells was associated with the decreased production of CDK7 mRNA and protein, which in turn, resulted in the reduction of CDK7 kinase activity in GA treated cells. The reduced CDK7 kinase activity is responsible for the inactivation of CDC2/p34 kinase and the G2/M phase cell-cycle arrest.


   Footnotes
 
{dagger}These authors contributed equally to this work. Back


   Acknowledgments
 
This work was supported by the National High Technology Research and Development Program of China (863 program, No.2002AA2Z3112, No.2004AA2Z3A10), National Natural Science Foundation of China (No. 30472044), Ministry of Education of China under Special Award for Critical Research (No. 104099), the National Natural Science Foundation of China (no. 30472044), and National Institutes of Health (SCORE S06GM-008205).

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received December 29, 2005; revised August 2, 2006; accepted August 13, 2006.


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D. Zhai, C. Jin, C.-w. Shiau, S. Kitada, A. C. Satterthwait, and J. C. Reed
Gambogic acid is an antagonist of antiapoptotic Bcl-2 family proteins
Mol. Cancer Ther., June 1, 2008; 7(6): 1639 - 1646.
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 Cancer Res.Home page
T. Yi, Z. Yi, S.-G. Cho, J. Luo, M. K. Pandey, B. B. Aggarwal, and M. Liu
Gambogic Acid Inhibits Angiogenesis and Prostate Tumor Growth by Suppressing Vascular Endothelial Growth Factor Receptor 2 Signaling
Cancer Res., March 15, 2008; 68(6): 1843 - 1850.
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 Clin. Cancer Res.Home page
S. Abbas, A. Bhoumik, R. Dahl, S. Vasile, S. Krajewski, N. D.P. Cosford, and Z. A. Ronai
Preclinical Studies of Celastrol and Acetyl Isogambogic Acid in Melanoma
Clin. Cancer Res., November 15, 2007; 13(22): 6769 - 6778.
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 BloodHome page
M. K. Pandey, B. Sung, K. S. Ahn, A. B. Kunnumakkara, M. M. Chaturvedi, and B. B. Aggarwal
Gambogic acid, a novel ligand for transferrin receptor, potentiates TNF-induced apoptosis through modulation of the nuclear factor-{kappa}B signaling pathway
Blood, November 15, 2007; 110(10): 3517 - 3525.
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 Proc. Natl. Acad. Sci. USAHome page
S.-W. Jang, M. Okada, I. Sayeed, G. Xiao, D. Stein, P. Jin, and K. Ye
Gambogic amide, a selective agonist for TrkA receptor that possesses robust neurotrophic activity, prevents neuronal cell death
PNAS, October 9, 2007; 104(41): 16329 - 16334.
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