Inhibitory Effects and Underlying Mechanism of 7-Hydroxyflavone Phosphate Ester in HeLa Cells

Chrysin and its phosphate ester have previously been shown to inhibit cell proliferation and induce apoptosis in Hela cells; however, the underlying mechanism remains to be characterized. In the present study, we therefore synthesized diethyl flavon-7-yl phosphate (FP, C19H19O6P) by a simplified Atheron-Todd reaction, and explored its anti-tumor characteristics and mechanisms. Cell proliferation, cell cycle progression and apoptosis were measured by MTS, flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling techniques, respectively in human cervical cancer HeLa cells treated with 7-hydroxyflavone (HF) and FP. p21, proliferating cell nuclear antigen (PCNA) and cAMP levels in Hela cells were analyzed by western blot and radioimmunoassay. Both HF and FP inhibited proliferation and induced apoptosis in HeLa cells via induction of PCNA/p21 expression, cleaved caspase-3/poly (ADP-ribose) polymerase (PARP)-1, elevation of cAMP levels, and cell cycle arrest with accumulation of cells in the G0/G1 fraction. The effects of FP were more potent than those of HF. The interactions of FP with Ca2+-calmodulin (CaM) and Ca2+-CaM-phosphodiesterase (PDE)1 were explored by electrospray ionization-mass spectrometry and fluorescence spectra. FP, but not HF, formed non-covalent complexes with Ca2+-CaM-PDE1, indicating that FP is an inhibitor of PDE1, and resulting in elevated cellular cAMP levels. It is possible that the elevated cAMP levels inhibit growth and induce apoptosis in Hela cells through induction of p21 and cleaved caspase-3/PARP-1 expression, and causing down-regulation of PCNA and cell cycle arrest with accumulation of cells in the G0/G1 and G2/M fractions. In conclusion, FP was shown to be a Ca2+-CaM-PDE inhibitor, which might account for its underlying anti-cancer mechanism in HeLa cells. These observations clearly demonstrate the special roles of phosphorylated flavonoids in biological processes, and suggest that FP might represent a potential new drug for the therapy of human cervical carcinoma.


Introduction
Among the variety of natural products, flavonoids have always attracted considerable interest because of their potential beneficial effects on human health and their widespread availability in fruits, vegetables, herbs and some beverages [1,2]. Most flavonoids have demonstrated anti-tumor properties including anti-proliferation, cell cycle arrest in G0/G1 or G2/M, and induction of differentiation and apoptosis in various cell lines [3][4][5].
A large number of phosphorus compounds possess P-O bond(s) as phosphate esters (e.g., DNA, RNA, ATP, phospholipids, etc.) and these esters of phosphoric acid play a vital role in many biological processes [6,7]. They appear to be synthesized and undergo interconversion with great ease in living organisms [8].
Our previous studies showed that phosphorylated flavonoids possess relatively stronger binding affinities towards proteins such as myoglobin, insulin, and lysozyme and more easily form noncovalent compounds with them, compared to non-phosphorylated forms [9,10]. As part of a screening program, we previously reported that phosphorylated chrysin did indeed exhibit stronger activity against HeLa tumor cells in vitro than non-phosphorylated chrysin [11]. These positive biomedical effects are mostly attributed to the potential of flavonoids to act as esters of phosphoric acid, but the underlying mechanism remains unclear. To explore these mechanisms, the phosphate ester (FP, C 19 H 19 O 6 P) of 7-hydroxyflavone (HF) was synthesized via a simplified Atheron-Todd reaction. It is an established fact that biochemical activities depend on the individual structure, and each compound needs to be studied systematically to assess its individual biological potency. In this study, we explored the anti-tumor characteristics of HF/FP with only one hydroxyl/ phosphorylated structure in the flavone subgroup. The effects of these compounds on proliferation and apoptosis in HeLa cells could thus be assessed and compared.
MTS, flow cytometry, proliferating cell nuclear antigen (PCNA) immunohistochemistry and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) techniques were used to gain an insight into the mechanisms of growth inhibition, cell proliferation, cell cycle progression, and apoptosis, respectively. Apoptosis was also determined by FACScanto II analysis. Semi-quantitative western immunoblotting was performed to assess the effects of HF/FP on protein expression levels. Alterations in cAMP levels were measured by radioimmunoassay (RIA). In addition, Ca 2+ -CaM-PDE complex inhibition was analyzed in vitro to provide detailed information about the possible mechanisms responsible for the anti-cancer activities. Moreover, we investigated the interactions between HF and FP, and the Ca 2+ -CaM-PDE enzyme system using fluorescence spectrometry to gain evidence for this mechanism in living systems. The results of this study may have important implications for the use of FP as a potent new agent for cancer prevention, as well as for other pharmacological and toxicological uses.   Figure 2D, .40 mM FP or 80 mM HF significantly increased the percentage of HeLa cells in G1 phase, accompanied by a decrease in the population in S phase, compared to the control group, suggesting that the cell cycle was arrested at G0/G1 phase. There was a significant increase in the cell population in G2/M phase (Figure 2 D and E) following treatment with FP, as well as a marked increase in the population in G0/G1 phase and a compensatory decrease in the population in S phase. These data suggest that HF induces cell cycle arrest in G0/G1 phase, while FP induces cell cycle arrest in both G0/G1 and G2/M phases.

FP-and HF-induced apoptosis
The TUNEL signal, as an apoptosis marker, appeared as a bluish-violet color, while the denser nuclei frequently moved towards the cell periphery. The percentage of apoptotic cells (% of apoptosis) in the control group was 7%, and this was increased to 22% in the HF group and up to 38% in the FP group after 48 h. There were a significant differences in apoptosis between the treated and control groups (p,0.05), as seen in Figure 3A and C. These results indicate that both FP and HF are potent inducers of apoptosis, but the effect of FP is stronger than that of HF.
To determine if cell death was accompanied by the development of an apoptotic or necrotic process, we further analyzed and quantified the phenotypic changes in apoptotic cells by double staining HeLa cells with Annexin V-FITC and PI. Cell apoptosis increased significantly after treatment with 10, 20, 40 and 80 mM FP/HF for various durations, compared to the control group ( Figure 3B, D and E). After treatment for 24 h, .40 mM FP could increase cell apoptosis, and 80 mM FP indirectly resulted in 89% apoptosis, whereas 80 mM HF only induced 12% apoptosis. In cells treated with 20 mM FP or HF for 48, 72 and 96 h, apoptosis induction was increased at 72 h, suggesting later stages of apoptosis in culture ( Figure 3B and E). As expected, cell death in the control group remained below 7%. These results are consistent with the results of the TUNEL method, further showing that HF and FP could induce apoptotic cell death in cervical cancer cells.

Effects of FP and HF on expression of PCNA in Hela cells
PCNA immunoreactivity (IR), represented by brownish-colored granules, was located mainly in the nuclei. Inactivated PCNA was located primarily in the cytoplasm, and translocated to the nuclei once activated. PCNA-IR intensities evaluated as the integration value (IV) after 48 h were 308 in the FP group and 348 in the HF group, which were significantly higher than the control group (512) (p,0.05) ( Figure 4). These results further showed that FP inhibited proliferation more intensively than HF, as demonstrated by the PCNA expression signal intensity in HeLa cells.
Effects of FP and HF on expression of p21/Waf1, caspase-3 and poly (ADP-ribose) polymerase (PARP) To determine the mechanisms responsible for cell cycle arrest and apoptosis by HF/FP, the protein expression levels of p21/ Waf1 (a cyclin-dependent kinase (CDK) inhibitor), and the apoptosis-related proteins cleaved caspase-3 and PARP-1 were examined using western blotting analyses. The expression levels of p21/Waf1 in the FP group were markedly increased in a timedependent fashion, compared to the control group ( Figure 5A and B), with more prominent expression after 48 h than after 24 h. p21/Waf1 expression in the FP group started to increase at 24 h, and became especially obvious at 48 h, whereas its expression in the HF group did not become apparent until 48 h, compared to the control group. Changes in p21/Waf1 expression in the FP treatment group were more marked (7-fold) in Hela cells, compared to that in the HF treatment group during the same period. This suggests that the up-regulation of p21/Waf1 might be related to the cell cycle arrest induced by HF and FP.
Levels of the apoptotic proteins, cleaved PARP-1 (90 kD) and caspase-3 (19 kD), were significantly elevated in FP-and HFcultured cells, compared to untreated control cells ( Figure 5 C and D). Cleaved caspase-3 started to increase at 24 h after FP treatment, and became especially obvious at 48 h and 72 h. While cleaved PARP-1 started to increase at 48 h after FP treatment, and became especially obvious at 72 h. In contrast, cleaved caspase-3 and PARP-1 became apparent after 72 h in the HF group. There were significant differences in cleaved-caspase 3/PARP-1 between the FP and HF groups. These results suggest that FP and HF induced HeLa cell injury or death by activating an apoptotic pathway involving impaired nuclear function and cellular homeostasis.

Effects of FP and HF on cAMP concentrations in Hela cells and PDE inhibition activity in vitro
Hela cells were pre-incubated in medium for 24 h and then incubated with 20 mM HF or FP at 37uC for a further 12 or 24 h. The concentrations of cAMP in the cells were measured in the various experimental groups ( Figure 6A). The cAMP concentrations were significantly increased to 12.260.67 pmol for HF and 16.260.87 pmol for FP at 12 h, and 15.260.83 (HF) and 24.661.68 (FP) pmol at 24 h, compared to control cells (9.160.78 pmol). These results represent increases of 134% and 178% at 12 h, and 167% and 270% at 24 h, respectively, which were significantly different from the levels in control cells (p,0.05, p,0.01). FP had a more significant effect on cAMP concentrations than HF.

Effects of FP on emission spectra of CaM-Ca2+-PDE system
The interaction between CaM and FP is shown in Figure 8A. When the FP concentration was increased, the emission peak of CaM decreased in each case, and the maximum emission wavelength increased from 330 to 350 nm.
The interaction between PDE and FP is shown in Figure 8B. The emission peak of PDE also decreased, accompanied by an increase in FP concentration, while the maximum emission wavelength increased from 335 to 360 nm. When equal concentrations of CaM and PDE were mixed with Ca 2+ , the emission intensity of the CaM-Ca 2+ -PDE system (l = 344 nm) increased significantly from 400 to 3,400 ( Figure 8C), indicating that the CaM-Ca 2+ -PDE enzyme system exhibited a strong intermolecular interaction. Figure 8D shows the emission spectra of the CaM-Ca 2+ -PDE system with increasing concentrations of FP; the emission intensity apparently decreased as the FP concentration increased, and the maximum emission wavelength increased simultaneously by 20 nm (from 340 to 360 nm). In contrast, the emission intensity of the CaM-Ca 2+ -PDE system decreased slightly as the HF concentration increased. According to the classical Stern-Volmer equation [12]: where F 0 is the emission intensity in the absence of quencher, F is the emission intensity in the presence of quencher, Kq is the quenching constant and [Q] is the quencher concentration.    system at 25uC is shown in Figure 8E. F 0 /F -[Q] lines at 25uC and 37uC, respectively, are shown in Figure 9A. The experiments demonstrated that a higher temperature was associated with a lower the slope of the quenching curve for the CaM-Ca 2+ -PDE system in presence of different amounts of FP. The combination of FP with CaM-Ca 2+ -PDE was a single static quenching process. The quenching data were therefore analyzed according to the equations Line weaver-Burk plots are shown in Figure 9B. The linearlydependent coefficients were 0.99 and 0.998. According to this equation, the binding constants at different temperatures could be calculated as k 37uC = 4.8610 4 L/mol, k 25uC = 6.24610 4 L/mol, respectively. These results showed that FP, but not HF, formed noncovalent complexes and showed high binding affinity with CaM-Ca 2+ -PDE.

Discussion
Most nontoxic dietary flavonoids are known to behave as general cell growth inhibitors in many kinds of cultured human cancer cell lines [13,14]. Many flavonoids can perturb cell cycle progression and induce G1 or G2/M cell cycle arrest, which is a fundamental activity in the process of cell proliferation [15,16]. The present results demonstrated that both HF and FP were able to significantly inhibit proliferation in human cervical cancer HeLa cells in dose-and time-dependent manners, with FP (IC 50 value, 18.5 mM at 72 h) having a much greater effect than HF (IC 50 value, 32.1 mM at 72 h). The present study also demonstrated a novel characteristic of FP in terms of the cell cycle, by observing that cell populations showed greatly decreased S phases and increased G0/G1 and G2/M phases compared to controls, indicating that FP controlled cell cycle progression at both the G1/ S and G2/M check-point transitions, whereas HF only arrested cell cycle progression at the G1/S check-point. The dose-and time-dependent effects on the cell cycle could contribute to the anti-proliferative effects of FP and HF.
Cell cycle arrest can lead to induction of apoptosis, and agents that modulate apoptosis affect the steady-state cell population, which is a useful means of increasing tumor-cell apoptosis for cancer therapy [17]. TUNEL labeling detected cells with typical apoptotic features after HF and FP treatment. The phenotypic changes characteristic of apoptosis were confirmed by cytometric analysis using double staining with FITC-Annexin V and PI. The   demonstrated increasing cell death of Hela cells after HF and FP uptake for 72 h, which represents a later stage of apoptosis. Thus, although both FP and HF could induce HeLa cell apoptosis, FP was more potent than HF in terms of activating apoptosis, indicating that FP may represent a potential anticancer agent for use in human cervical cancer therapy. Cell growth is controlled by the balance between proliferation and programmed cell death (apoptosis). In order to further understand the molecular mechanisms responsible for the anticancer activities of HF and FP, the expression levels p21/Waf1, PCNA and apoptosis-related proteins were examined. p21/Waf1 inhibits CDK activities and prevents cell cycle progression [18,19]. The development of sporadic tumors is generally associated with reduced expression of p21/Waf1 [20]. Moreover, increased expression of p21/Waf1 has been demonstrated to inhibit proliferation and promote apoptosis of malignant cells in vitro and in vivo [21,22]. PCNA is markedly expressed in proliferating cells and in most malignant tumor cells, and is therefore used as a proliferative/malignancy biomarker in cancer [23,24]. Cleaved caspase-3 has been verified as an activated form of caspase-3 that acts as a lethal protease at the most distal stage of the apoptotic pathway [25]. At the onset of apoptosis, caspase-3 proteolytically cleaves PARP, which is a nuclear DNA-binding zinc-finger protein that influences DNA repair, DNA replication, modulation of chromatin structure, and apoptosis [26].
We investigated the involvements of p21/Waf1 and PCNA proteins in the antiproliferative effects of HF-and FP in HeLa cells. FP induced cell cycle arrest in both G0/G1 and G2/M phases as shown by up-regulation of p21 and down-regulation of PCNA in HeLa cells. Enhanced expression of cleaved caspase-3 and PARP-1 (90 kD) in FP-or HF-treated HeLa cells at 48 h provided further evidence for promotion of apoptosis by FP and HF. However, the levels of cleaved PARP-1 were lower than those of cleaved caspase-3 after FP and HF treatment, indicating that activated caspase-3 may activate other downstream caspase family effectors. FP is characterized by the presence of a 7-hydroxyflavone basic ring skeleton, with an extra phosphate ester substitution. This structural difference is related to its mechanism of action and potency. Together, FP-induced apoptosis and proliferation inhibition contribute directly to up-regulation of p21/Waf1, decreased PCNA, and increased cleavage of caspase-3 and PARP-1. In contrast, 7-hydroxyflavones lacking this phosphate ester substitution mediated cell cycle arrest in G0/G1 phase and showed antiproliferative activity consistent with inhibition of PCNA activity. The induction of p21/Waf1 by HF might be responsible for cell-cycle arrest but not apoptosis (elevated p21 vs. no obvious apoptosis for HF at 48 h). These results suggest that FP and HF might induce growth inhibition and apoptosis by different mechanisms.
cAMP signaling pathways are known to provide major routes for controlling cellular function [27,28]. In an in vitro experiment, cAMP was considered to exert a growth-inhibitory action on SKOV cells via induction of p21 expression and cell cycle arrest, with accumulation of cells in the G0/G1 fraction of the cell cycle [29]. In the present study, the FP-mediated activation of p21/ Waf1 in Hela cells elicited a profound increase in cAMP concentrations, which appear to inhibit cell growth through induction of p21 expression and inhibition of PCNA and cell cycle progression in G1 or G2/M. cAMP inactivation is known to occur through hydrolysis to 59-AMP, which is achieved by cAMP PDE activity [30,31]. Within the PDE enzyme family, PDE1 activities are stimulated by Ca 2+ and CaM, and can specifically control cAMP degradation within the cell regulatory system. In addition, these isoenzymes may be inhibited by several flavonoids [32]. As previously reported for a number of non-selective PDE inhibitors, the current results showed that FP preferentially inhibited CaMactivated PDE1 (which hydrolyzes cAMP) isolated from bovine brain, with an IC 50 value of 22.3 mM, which was significantly lower than the IC 50 (43.5 mM) necessary for inhibition of basal PDE, indicating that FP also interacts with CaM. Both HF and FP may thus act as PDE inhibitors with different activities, resulting in increased levels of cyclic nucleotides. It is thus likely that the effect of FP on p21 regulation may be associated with an increase in intracellular cyclic nucleotide levels (via PDE inhibition). FP showed greater potency than HF in most of the current experiments, indicating that the presence of a phosphate ester in the chemical structure of FP (7-position) increases its pharmacological activity by producing a spatial conformation that favors interaction with PDE and other potential cellular targets.
Comprehensive studies on the nature of the interactions between small molecules and proteins are of fundamental importance in the biomedical and pharmaceutical sciences. In this context, we extended the scope of this study to examine the interaction between FP and CaM-Ca 2+ -PDE1 using fluorescence spectrometry combined with ESI-MS. A new noncovalent complex (FP-CaM-Ca 2+ ) analyzed by ESI-MS further verified the stronger binding affinity of FP with CaM-Ca 2+ , compared to HF. Furthermore, the FP-CaM-Ca 2+ noncovalent complex was likely to influence PDE1 species activities via specific CaM-Ca 2+ stimulation. The fluorescence intensity of a mixed solution of CaM-Ca 2+ and PDE in vitro was also significantly elevated compared to either CaM-Ca 2+ or PDE1 alone, confirming that specific induction by Ca 2+ /CaM stimulated PDE1 activity. The effects of FP on the emission spectra of the CaM-Ca 2+ -PDE protein-enzyme complex system demonstrated an enhanced molecular interaction between FP and the CaM-Ca 2+ -PDE enzyme complex system.
Determination of the FP and CaM-Ca 2+ -PDE1 binding constants provided information on the binding sites and the main acting forces in the studied complexes. The binding effects detected by fluorescence and ESI-MS assays were in agreement with the results of PDE activity, despite the different principles of the methods. The results obtained could facilitate our understanding of the physiological effects by focusing on the structural features. Our previous study showed that the individual biological potency of the phosphate ester of chrysin was related at least partly to its phosporylated structure [11]. Phosphorylated flavones might thus combine with the active site of PDEs by H-bond interactions, preventing the CaM-Ca 2+ -PDE enzyme complex from effectively hydrolyzing cAMP.
To summarize, this study demonstrated that FP has clear anticancer effects in Hela cells, probably mediated by an increase in cytosolic cAMP via inhibition of PDE. FP exerts growth inhibitory and apoptosis-inducing actions through induction of p21, caspase-3 and PARP-1, and down-regulation of PCNA associated with cell cycle arrest. In contrast, HF has lower activity against HeLa tumor cells under the same conditions. These observations clearly demonstrate the importance of phosphorylated flavonoids in biological processes, and suggest that FP might have promising potential as a new anti-cancer drug.

MTS assay
The test compounds (HF and FP) were dissolved in dimethyl sulfoxide (DMSO) #0.1%. HeLa cells (human cervical carcinoma, Shanghai Institutes of Biological Sciences) were cultured in DMEM (Gibco, Invitrogen, USA) supplemented with 10% fetal bovine serum (Gibco). Cell viability was measured in the presence or absence of HF or FP using a cell viability assay, as previously described [33]. The cells were harvested in the log phase of growth and inoculated onto 96-well plates at a final concentration of 3610 3 cells per well. After 24 h of incubation at 37uC under 5% CO 2 , the cell cultures were treated with FP/HF at 5, 10, 20, 40, 80 and 160 mM. [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2(4-sulfophenyl)-2H-tetrazolium/phenazine methosulfate (MTS/PMS; Promega, Madison, WI, USA) 20 ml was added to each well, and the absorbance at 490 nm was measured according to the manufacturer's recommendations. The mean value was obtained from four replicate wells after exposure to the agents for 24, 48, 72 and 96 h, respectively. A control group treated with no any agents was performed simultaneously. The growth inhibition ratio was calculated according to the following equation: inhibition ratio (%) = (control group2treated group)/control group6100%.
Preparation of specimens 20 mM HF and FP were added to HeLa cell cultures for 24, 48 and 72 h. TUNEL, PCNA immunocytochemistry tests and western blotting (p21/Waf1, caspase-3 and PARP1) were performed, based on the above MTS assay. Experimental and control specimens for TUNEL and immunocytochemistry assays were fixed on slides with 4% paraformaldehyde for 20 min at room temperature, followed by washing in phosphate-buffered saline (PBS), and storage at 220uC.

Cell-cycle analysis
HeLa cells were seeded at a density of 1610 5 onto 6-well plates and incubated for 24, 48, 72 and 96 h in the absence or presence of the test compounds (10,20,40 and 80 mM concentrations after reaching 70% confluency. The cells were washed twice with PBS, then harvested by trypsinization and washed twice with PBS. Then pellets were resuspended and fixed in 70% alcohol for 1 h at 4uC. The suspension was then centrifuged in a microfuge for 5 min at 800 rpm, and the pellet was resuspended in PBS containing PI (50 mg/ml), Triton X-100 (0.1%, v/v) and RNase A (100 mg/ml). After 30 min incubation, the cell cycle distribution was examined using a FACScano II SE flow cytometer with Modfit software (Becton Dickinson, San Jose, CA).
TUNEL method for detection of apoptosis TUNEL analysis to detect apoptosis was performed as described previously, with 1 ml terminal dUTP-transferase (TdT, Promega, Madison, WI, USA) and 1 ml biotin-16-dUTP (Promega) [11]. Positive bluish-violet signals were visualized using streptavidinalkaline phosphatase reaction with 5-bromo-4-chloro-3-indolylphosphate and nitroblue tetrazolium (BCIP-NBT, Promega) as the substrate. TdT was omitted from the buffer in the negative control specimen. The percentage of apoptosis (% of apoptosis) according to the positive signal was calculated for each slide, in which ten fields of view were selected under an oil-emersion microscope (Nikon, TE2000-U, Japan). Apoptosis (%) = (number of apoptosis cells/total number of tumor cells)6100. Annexin V-FITC/PI double staining for apoptosis by flow cytometry HeLa cells were seeded at a density of 5610 5 onto 6-well plates and incubated for 24, 48, 72 and 96 h in the presence or absence of the test compounds (10,20,40 and 80 mM). Apoptosis assay was performed with an Apoptosis Assay Kit (Baosai Biology, Beijing, China) using a flow cytometer, following the manufacturer's instructions. Briefly, cells were trypsinized and washed with PBS. The pellets were resuspended in PBS to a concentration of 5610 5 cells. The cells were then incubated with 10 ml FITC-Annexin-V dissolved in 300 ml binding buffer in the dark at room temperature for 15 min. The pellets were stained with 5 ml PI in 200 ml binding buffer. Emission wavelengths of 518 nm (Annexin V-FITC) and 617 nm (PI) were used to identify the populations of viable cells (Annexin V 2 PI 2 ), early apoptotic cells (Annexin V + PI 2 ), necrotic cells (Annexin V 2 PI + ), and late apoptotic cells (Annexin V + PI + ). The collected events per sample were 10,000. Apoptotic percentage (AP %) = (upper right quadrant (UR)+lower right quadrant (LR)) %.

Immunocytochemistry
Paraformaldehyde-fixed slides were treated with 0.5% Triton X-100/PBS solution for 30 min followed by 3% H 2 O 2 for 5 min. The slides were heat-renatured in 0.05 mM citric acid buffer (pH 6.0 for antigen retrieval), and blocked with 1:10 diluted normal goat serum in PBS for 30 min. After removal of excess serum, slides were incubated overnight at 4uC with monoclonal antibody against PCNA (Zymed, Invitrogen, USA) freshly diluted at 1:100, followed by incubation with biotin-labeled goat antimouse serum and streptavidin conjugated peroxidase at 37uC for 20 min. 3, 39diaminobenzidine 0.05% and 0.01% H 2 O 2 were used as substrates to develop a brownish positive IR signal. PBS was substituted for the specific primary antibody in the negative control. Slides were examined and 200 cells were counted in ten different fields of view by two independent, blinded observers, using an oil-emersion microscope. The IV of PCNA-IR was based on a previously reported method [34,35], and modified as follows: 0 = negative (2, no appreciable staining in cells); 1 = weak positive (+, readily-appreciable brown staining distinctly marking the tumor cell cytoplasm and/or nucleus); 2 = medium positive (++, dark brown staining in tumor cells obscuring the cytoplasm and/ or nucleus); 3 = strong positive (+++, very strong staining of nucleus and/or cytoplasm). The IV of PCNA-IR was scored semiquantitatively by multiplying the number of positive cells (0-200) by the staining intensity (grade 1-3), thus producing a score of 0-600.
Western blotting analysis of caspase-3, PARP-1 and p21/ waf1 proteins in Hela cells HeLa cells were seeded in 24-well plates (5610 4 /well) and cultured for 24 h. HF or FP 20 mM were added to the cells for a further 24 or 48 h. Extracts from each group cells were prepared and western immunoblotting analysis was performed as follows: The cells were washed twice with PBS, scraped from the culture dishes, and treated for 20 min on ice with lysis buffer containing 1 mM EGTA, 1 mM EDTA, 150 mM NaCl, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM phenylmethyl-sulfonyl fluoride, 1 mM NaVO 4 and 1 mg/ml leupeptin, 1 mg/ml aprotinin and 20 mM HEPES-KOH (pH 7.9). Cell lysates were centrifuged at 13,000 rpm for 15 min at 4uC. Total protein concentrations were determined and equal amounts of proteins in each group were then separated by 12% sodium dodecyl sulfatepolyacrylamide gel electrophoresis. The gels were transferred to a nitrocellulose membrane (Amersham, GE Heathcare, USA) using a semi-dry electroblotting system (Bio-Rad, USA). The membranes were blocked with 3% (w/v) bovine serum albumin in PBS with 0.2% (v/v) Tween-20 (PBST) for 1 h, and then incubated with the primary mouse monoclonal antibody (anti-caspase3 and anti-PARP-1, Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1:200), mouse monoclonal antibody (against p21 Waf1 , Santa Cruz, 1:200), and b-actin monoclonal antibodies (1:1,000; Santa Cruz) at 4uC overnight with rocking. Membranes were rinsed three times with PBST, and incubated with secondary goat anti-mouse IgG conjugated with IRDye700 fluorescence in (1:10,000 dilution; LI-COR, USA) for 1 h at room temperature. Membranes were rinsed twice with PBST and once with PBS. Subsequent analysis was performed using a Li-COR Odyssey system and quantified using Odyssey infrared imaging software, with b-actin as a loading control.

RIA examination of cAMP in cells
HeLa cells were seeded at a density of 3610 4 onto 24-well plates and incubated for 12 or 24 h in the presence or absence of the test compounds (20 mM). Cells were washed three times with PBS and then freeze-thawed twice with 0.5 ml cold HCl (50 mM) at 280uC. Cell lysates were centrifuged at 13,000 g for 10 min at 4uC. The supernatants were freeze-dried and stored at 280uC. The cAMP concentration of each sample was measured using commercial RIA kits (Shanghai University of Traditional Chinese Medicine, Shanghai, China). The minimum detection limit for cAMP was 0.1 pmol/ml for nonacetylated samples; cross-reaction with cGMP was less than 0.001%. In brief, duplicate aliquots of samples (100 ml) and 100 ml of each point of cAMP standard curve (20,10, 5, 2.5, and 1.25 pmol/ml) were dispensed into conical tubes containing 100 ml of assay buffer, then incubated with 100 ml of 125 I-cAMP (25,000 cpm) and 100 ml of diluted primary antibody (rabbit anti-cAMP). The initial dilution of the antiserum was 1:500. The maximum binding (B 0 ) was determined by replacing standard cAMP by 100 ml of assay buffer. The nonspecific binding (NSB) tubes contained 200 ml of assay buffer and 100 ml of 125 I-cAMP. Total count tubes (Tc) contained 100 ml of 125 I-cAMP. The following day, bound and free fractions were separated by adding 100 ml of second-antibody solution (0.05% v:v normal rabbit serum, 0.45% v:v sheep anti-rabbit anti serum,0.4% w:v BSA, 0.05% w:v microcrystalline cellulose, 0.5%w:v polyethylene glycol 6000 in phosphate buffer) to all except the Tc tubes, followed by further incubation (1.5 h) at room temperature. The tubes were then washed with 2 ml of phosphate-BSA-Tween 20 TM buffer and centrifuged at 2,500 g at 4uC for 30 min. The supernatant was discarded and the pellet was washed again. The radioactivity was measured in a gamma counter with an efficiency of 75%.

Determination of PDE activity
PDE activity was measured using the PDE-Glo TM Assay (Promega, Madison, WI, USA), according to the manufacturer's instructions. HF and FP were serially titrated manually at 1:2, and added to the assay plate containing bovine brain PDE (Sigma-Aldrich Cat.# P9529) or CaM-activated PDE, in which 2 U CaM (Sigma-Aldrich Cat.# P4874) were incubated with 0.015 U PDE and 0.03 mM Ca 2+ for 30 min, and pre-incubated for 5 min before substrate addition. cAMP substrate 2 mM was added to each well for 5 min. Luminescence was recorded using a GloMaxH-Multi Microplate Reader (Promega). Value was expressed as a relative light units (RLU). The IC 50 values were determined by non-linear regression analysis by fitting to hyperbolic inhibition. The maximal final concentration of DMSO in the control samples had no effect on PDE activity. Because of the limited solubility of the tested flavonoids, the highest concentration used in these experiments was 200 mM.

Mass spectrometry conditions
A mass spectrometer (Bruker-Esquire 3000 Ion Trap, Bruker Daltonics, Bermen, Germany) installed with an ion-spray source working in the positive ion mode was employed. The neutralizer pressure was 12 psi, the dry gas flow rate was 9.00 l/min, and the capillary voltage was held at 4 kV. The rolling average was seven. The ion count cumulative (ICC) was set at 30,000. Dissolved samples were continuously infused into the ESI chamber at a flow rate of 4 ml/min using a 744900 syringe pump (Cole-Parmer Instrument Co., USA).

Fluorescence spectra
The binding reactions between FP and Ca 2+ -CaM-PDE enzyme system in the aqueous phase were studied using fluorescence spectrometry. Fluorescence spectra were detected using a F4500 spectrofluorometer with an excitation wavelength of 280 nm and an emission range set between 290 nm and 450 nm. The excitation wavelength for proteins is generally about 280 nm, and this wavelength was therefore selected to study the FP and Ca 2+ -CaM-PDE enzyme system interaction. No fluorescence was emitted by FP under this excitation wavelength. The quenching experiments were repeated with different quantities of FP at 25uC and 37uC, for the same Ca 2+ -CaM-PDE enzyme concentration.

Statistical analysis
At least three independent experiments were carried out for each variable. Data are given as mean 6 SE. The statistical analysis was made with SSPS10.0. Differences (p,0.05), indicated by asterisks, were considered statistically significant.