2-Methoxyestradiol, an Estradiol Metabolite, Inhibits Neointima Formation and Smooth Muscle Cell Growth via Double Blockade of the Cell Cycle
2-Methoxyestradiol (2-ME), an endogenous metabolite of estradiol with no affinity for estrogen receptors, is a potent anticarcinogenic agent (in phase II clinical trials) and mediates the inhibitory effects of estradiol on smooth muscle cell (SMC) growth. Here we studied the intracellular mechanisms by which 2-ME inhibits SMC growth and whether 2-ME prevents injury-induced neointima formation. 2-ME concentrations that inhibit proliferation of cycling human aortic SMCs by ≥50% blocked cell-cycle progression in G0/G1 and in G2/M phase, as determined by flow cytometry. Consistent with the cell-cycle effects, at a molecular level (Western blots), 2-ME inhibited cyclin D1 and cyclin B1 expression; cyclin-dependent kinase (cdk)-1 and cdk-2 activity; and retinoblastoma protein (pRb), extracellular signal-regulated kinase (ERK) 1/2, and Akt phosphorylation. 2-ME also upregulated the Cdk inhibitor p27 and interfered with tubulin polymerization. Moreover, 2-ME augmented COX-2 expression, suggesting that it may also inhibit SMC growth via prostaglandin formation. In rats, treatment with 2-ME abrogated injury-induced neointima formation; decreased proliferating SMCs; downregulated expression of proliferating-cell nuclear antigen (PCNA), c-myc, cyclin D1, cyclin B1, phosphorylated Akt, phosphorylated ERK1/2, p21, and pRb; inhibited cdk-1 and cdk-4 activity; and upregulated expression of cyclooxygenase (COX)-2 and p27. Caspase-3 cleavage assay and fluorescence-activated cell-sorting (FACS) analysis showed no evidence of apoptosis in 2-ME-treated SMCs, and TUNEL staining in carotid segments showed no evidence of 2-ME-induced apoptosis in vivo. The antimitotic effects of 2-ME on SMCs are mediated by the inhibition of key cell-cycle regulatory proteins and effects on tubulin polymerization and COX-2 upregulation. These effects of 2-ME most likely contribute to the antivasoocclusive actions of this endogenous compound.
2-Methoxyestradiol (2-ME), a metabolite of estradiol with no affinity for estrogen receptors (ERs),1 inhibits tumor growth,2 neovascularization, and growth of cancer cells (angiosarcoma, gastric, cervical, colorectal, hepatocellular, prostate, lung, pancreatic, breast, neuroplastoma, leukemia, multiple myeloma).2 Based on these findings, 2-ME is in phase II clinical trials for cancer.3
Antimitotic therapies used in cancer may also protect against vascular disorders4 because abnormal growth of vascular smooth muscles (SMCs) contributes to vascular remodeling, such as neointima formation, atherosclerosis, and injury-induced restenosis.1 Therefore, drugs capable of inhibiting SMC growth by targeting key mitogenic mechanisms are effective in protecting against cardiovascular disease.1
Because 2-ME is an anticancer drug, it too may have therapeutic potential in preventing vasoocclusive diseases linked to proliferative disorders such as restenosis after balloon angioplasty and bypass vein graft disease. This hypothesis is strengthened by our recent findings that 2-ME is a potent inhibitor of SMC proliferation and migration and extracellular matrix production1 and that sequential metabolism of estradiol to methoxyestradiols, such as 2-ME, plays a major role in mediating the inhibitory effects of estradiol on SMC growth.1 Hence, 1 of the aims of the present study was to test the hypothesis that 2-ME can inhibit vascular injury-induced neointima formation.
Although 2-ME is a potent antimitogenic agent, the mechanisms by which 2-ME inhibits SMC growth are unknown. Hence another aim of this study was to clarify the mechanisms by which 2-ME mediates its antimitogenic effects on human aortic SMCs. Specifically, we investigated the effects of 2-ME on cell-cycle progression/distribution, cell morphology, the expression of key proteins responsible for regulating cell-cycle progression from G0 to M phase (such as cyclin D1, cyclin B1, and cyclin-dependent kinase [cdk]-1 and cdk-4) and the phosphorylation (activation) of important signal transduction molecules involved in cell-cycle regulation (such as extracellular signal-regulated kinase [ERK] 1/2, Akt, and retinoblastoma protein [pRb]).
Similar to taxol, colchicine, and paclitaxel, 2-ME is known to interfere with the dynamics of tubulin polymerization,5 and tubulin interfering agents are known to increase the expression of cyclooxygenase-2 (COX-2) and thereby enhance the biosynthesis of prostaglandins.5 Because some prostaglandins inhibit SMC growth,6 it is conceivable that this mechanism also participates in the pharmacological actions of 2-ME in the blood vessel wall. Accordingly, a third aim of this project was to test the hypothesis that 2-ME increases COX-2 expression in SMCs.
Materials and Methods
Human (female) aortic smooth muscle cells (HASMCs; 6th to 8th passage; Cascade Biologics Inc) were cultured under standard tissue culture conditions (37°C, 5% CO2) in M231 culture medium containing SMC growth supplement (Cascade Biology Inc, Switzerland) and as described previously.7
Effects of 2-ME on SMC Growth, Cell-Cycle Distribution Analysis, and Intracellular Mechanisms
For details, see the online data supplement, available at http://circres.ahajournals.org.
HASMCs at 60% confluence were treated with different concentrations (0 to 10 μmol/L) of 2-ME for various time periods. For cell-cycle analysis, cells were stained with propidium iodide (PI), and DNA content was analyzed by flow cytometry. In some experiments, cells were starved for 48 hours in serum-free DMEM/F12 before treatment with 2-ME (0 to 10 μmol/L) for different time periods. To assess whether 2-ME inhibits the transition of SMCs from G2/M to G0/G1 phase of the cell cycle, bromodeoxyuridine (BrdU) pulse-chase assays were conducted in SMCs treated with 2-ME for 24 hours.
Changes in the phosphorylation state and activity of signal transduction proteins and changes in the expression of cell-cycle regulatory proteins and COX-2 were analyzed by Western blots or labeling with radioactive γ-P32-ATP. The influence of 2-ME on the dynamics of tubulin polymerization was assayed by immunofluorescence microscopy and by Western blots following separation of free and polymerized tubulin.
Carotid Artery Injury Studies
For details, see the online data supplement.
Balloon injury-induced neointima formation was assessed in animals (male Wistar Kyoto rats; 350 to 400 g; RCC, Fullinsdorf, Switzerland), as described previously.8 Osmotic pumps (ALZET minipumps) containing vehicle or 2-ME were implanted for intravenous delivery of vehicle or drug (350 μg/kg per day). After 14 days, the animals were euthanized and perfusion fixed for morphometric analysis.8 Before euthanasia, blood samples were drawn for red blood cell (RBC) count, white blood cell (WBC) count, hematocrit, testosterone levels, and 2-ME levels by gas chromatography-mass spectroscopy (GC-MS). Also, testicles were removed and weighed.
To assess the impact of 2-ME on proliferation of intimal SMCs following balloon injury, animals (placebo n=7, treated n=7) receiving the vehicle or 2-ME were euthanized and perfusion fixed 7 days after balloon injury, when the proliferative activity of SMCs peak. Sections were immunostained with Ki67 to assess proliferating SMCs and with terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) to assess apoptotic cells. To assess the effects of 2-ME on cell-cycle and signal-transduction proteins in vivo, rats (placebo, n=3; treated, n=3) were euthanized on day 8 and the carotid arteries snap frozen in liquid nitrogen. Subsequently segments from placebo or 2-ME-treated animals were homogenized and lysed and various proteins analyzed using Western blotting.
Treatment effects on cross-sectional areas were analyzed by using ANOVA or the nonparametric Kruskal-Wallis test. Expression and growth data were analyzed using ANOVA, and statistical significance (P<0.05) was calculated using Fisher’s least-significant difference test. Unless specified, data for cell culture experiments represent 3 experiments in triplicates using separate cultures.
2-ME Inhibits Cell Growth in Proliferating HASMCs and Changes Cell Morphology
In cycling SMCs treated with 0 to 10 μmol/L of 2-ME, a concentration- and time-dependent inhibition of cell proliferation was observed (Figure 1A). A concentration-dependent accumulation of cells in the G2/M phase (from 16.0±0.6% in untreated to 40.2±0.8% in cells treated with 5 μmol/L of 2-ME) and a parallel decrease in the G0/G1 phase was observed after 48 hours of treatment (from 73.4±0.8% in untreated to 52.2±0.4% in cells treated with 5 μmol/L 2-ME; Figure 1B). These effects did not further change until day 6, and the cell population was equally distributed between G0/G1 and G2/M phases (Figure 1C). In SMCs pulse labeled with BrdU and treated with 2-ME for 24 hours, the ratio of BrdU-labeled SMCs in G0/G1 to G2/M decreased significantly (Figure 1D), suggesting that the BrdU-positive cells accumulated in G2/M and therefore that 2-ME treatment inhibited G2/M to G0/G1 transition.
To elucidate whether stabilization of the G0/G1 cell population resulted from inhibition of the progression into S phase, HASMCs were made quiescent by serum starvation for 48 hours and then stimulated with 5% serum to enter S phase in the presence or absence of 2-ME (2 μmol/L). Subconfluent HASMCs accumulated in G0/G1 after serum starvation (78.9±0.4% in G0/G1 phase, 5.8±0.5% in S phase and 15.2±0.1% in G2/M). Following 24 hours of serum treatment, the population of G0/G1 cells significantly decreased to 63.4±1.7%, with a concomitant increase of SMCs in S phase (17.9±0.8%) and a minor increase in G2/M phase (18.7±0.9%). 2-ME significantly (P<0.05) inhibited G1→S progression following serum stimulation, as reflected by the higher percentage of cells remaining in G0/G1 phase (67.2±0.2%; P<0.05 versus serum alone) and the lower percentage of cells entering S phase (14.2±0.3%; P<0.05 versus serum alone).
To investigate the mechanisms by which 2-ME induced G0/G1- and G2/M-phase arrest, we analyzed the expression of proteins regulating the cell-cycle progression at these phases. Treatment of SMCs with 2-ME attenuated the phosphorylation of pRb and decreased cyclin D1 expression in a concentration- and time-dependent manner (Figure 2A and 2B). Moreover, 2-ME abrogated ERK1/2 phosphorylation in a time- and concentration-dependent fashion (Figure 2C). Similar to ERK1/2, 2-ME inhibited Akt phosphorylation in a concentration-dependent manner (Figure 2D). Treatment of SMCs with 2-ME also caused a concentration-dependent reduction in cyclin B1 expression (Figure 2D). Treatment of SMCs for 48 hours with 2-ME inhibited cdk-4 and cdk-1 activities associated with cyclin D1 and cyclin B1, respectively, as measured by Western blotting or radioactive assay using γ-P32-ATP (Figure 3A and 3B).
Treatment with 2-ME induced protein levels of the cdk inhibitor p27 in a concentration-dependent manner (Figure 4A). In contrast, the expression of the cdk inhibitor p21 was downregulated under the same treatment conditions (Figure 4B). Liu et al9 provided evidence that p21 expression is positively regulated by ERK1/2; hence, the inhibitory effect of 2-ME on ERK1/2 phosphorylation may potentially explain the downregulation of p21. To test this possibility, we treated cells with the specific ERK1/2 inhibitor PD98059 (10 μmol/L) and measured p21 expression. Similar to 2-ME, PD98059 significantly (P<0.05) inhibited p21 protein levels (Figure 4C). PD98059 specifically inhibited ERK1/2 phosphorylation with no effect on Akt (Figure 4C). These data provide evidence that the inhibitory effect of 2-ME on ERK1/2 phosphorylation is responsible for downregulating p21 expression.
In SMCs treated with 2-ME (2 μmol/L), the morphology changed from elongated spindle-shaped cells to rounded cells, with a significant reduction in cell size (Figure 5A). The 2-ME effect was not attributable to toxicity, as SMC shape reversed when the treatment was withdrawn (data not shown). Immunofluorescent staining of SMCs with α-tubulin showed a concentration-dependent inhibitory effect of 2-ME on tubulin polmerization (Figure 5B). Western blot analysis of polymerized and free tubulin fractionated from SMCs treated for 48 hours with 0 to 10 μmol/L 2-ME showed a concentration-dependent inhibition in the amount of polymerized tubulin (Figure 5C). Similar inhibitory effects were observed in SMCs treated with colcemid (0.1 μg/mL), a positive control.
COX-2 plays an important role in regulating SMC growth6 and mediates in part the protective actions of estrogens on atherosclerosis.6 Because tubulin-interfering agents modulate COX-2 activity,5 we assessed the effects of 2-ME on COX-2 expression. Treatment of SMCs with 2-ME induced COX-2 expression in a concentration-dependent manner (Figure 5D). Moreover, this activation was accompanied with a parallel decrease in ERK1/2 phosphorylation (Figure 3A), which is known to regulate COX-2 activity.5 To assess whether upregulation of COX-2 contributes to the antimitogenic actions of 2-ME, the effects of 2-ME on FCS-induced SMC growth was analyzed in the presence and absence of NS-398, a specific COX-2 inhibitor. As shown in Figure 5D, the concentration-dependent inhibitory curve of 2-ME was shifted to the right, suggesting that COX-2 contributes to the inhibitory actions of 2-ME and that other pathways also play an important role.
Effects of 2-ME on Neointima Formation
Morphometric analysis of the carotid arteries showed significant intimal thickening following balloon injury, and this was significantly inhibited in rats receiving 2-ME for 14 days (Figure 6A). Compared with placebo group (n=12; intima 0.116±0.011 mm2), the neointima formation was reduced by 46% in rats receiving 2-ME (n=10; 0.0626±0.0069 mm2; P<0.02 versus placebo) (Figure 6A). The media and lumen areas did not significantly differ between the placebo and the 2-ME groups (media: 0.08±0.005 mm2 in placebo group and 0.072±0.002 mm2 in 2-ME group; lumen: 0.293±0.039 mm2 in placebo group and 0.2756±0.0226 mm2 in 2-ME group); however, the intimal/medial ratio was significantly reduced in animals receiving 2-ME (1.446±0.04 in placebo group versus 0.8718±0.023 in 2-ME group; P<0.05 versus placebo; see the online data supplement). In carotid arteries obtained from animals receiving placebo, proliferating Ki67-positive cells were observed (Figure 6B). As compared with the placebo group, a significant decrease in Ki67-positive SMCs were observed in arteries obtained from animals treated with 2-ME (Figure 6B). Rats treated with 2-ME lost body weight (365±2 g in placebo group versus 317±4 g in 2-ME group; 16% reduction; P<0.05; see the online data supplement); however, treatment with 2-ME was not associated with any toxic adverse effects. In this regard, the proliferation (Ki67-positive cells) of duodenal epithelial cells was not influenced by 2-ME treatment (see the online data supplement). Moreover, the testis weight, testosterone levels, WBC count, RBC count, and hematocrit did not differ between placebo and 2-ME groups and were, respectively: 1.97±0.17 versus 1.95±0.13 g; 4.2±0.4 versus 4.0±1.2 ng/mL; 4.661±0.451×103/mm3 versus 4.415±0.509×103/mm3; 6.833±0.336×103/mm3 versus 7.137± 0.429×103/mm3; and 40.89±1.535% versus 39.50±1.509%. (See the online data supplement.)
To assess whether the inhibitory effect of 2-ME on intimal growth following balloon injury was associated with changes in the expression of proteins regulating SMC growth, we analyzed key signal transduction and cell-cycle proteins in carotid arteries. As shown in Figure 7A, compared with placebo, treatment with 2-ME downregulated the expression of proliferating cell nuclear antigen (PCNA) and c-myc by 74% and 58%, respectively, indicating that 2-ME inhibited cell growth. Additionally, 2-ME downregulated the expression of cyclin D1, cyclin B1, phosphorylated ERK1/2, and phosphorylated Akt by 54%, 77%, 78%, and 86%, respectively, and inhibited phosphorylation of pRb. 2-ME upregulated the expression of p27 and COX-2 by 380% and 294%, respectively. Similar to cultured SMCs, 2-ME downregulated the expression p21 by 62% in injured vessels. Moreover, 2-ME significantly inhibited (P<0.05 versus placebo) cdk-1 and cdk-4 activities by 43% and 52%, respectively, in injured vessels (Figure 7B).
Effects of 2-ME on Apoptosis
To assess whether apoptosis contributed to the growth inhibitory effects of 2-ME, we assayed the effects of 2-ME versus H2O2 (positive control) on apoptosis. In SMCs, H2O2 (50 mmol/L), but not 2-ME (2 μmol/L), induced a sub-G1 cell population (Figure 8A) and caused caspase-3 fragmentation and β-actin degradation (Figure 8B), suggesting that the inhibitory effects of 2-ME on SMC growth were independent of apoptosis. Indeed, in SMCs treated with 2-ME, the cell number did not fall below the cell number at treatment initiation. Also, microscopic observation showed no evidence of apoptotic body formation. Moreover, in carotid segments immunostained for TUNEL-positive apoptotic cells, we observed very few TUNEL-positive cells, and their numbers did not significantly differ in vessels between placebo- and 2-ME-treated animals (Figure 8C).
Here we provide the first evidence that 2-ME protects against injury-induced neointima formation in rats and arrests growth of human aortic SMCs via double blockade of the cell cycle. We demonstrate that 2-ME: (1) arrests proliferating SMCs in G0/G1 and G2/M-phases; (2) inhibits the signaling pathways ERK1/2 and Akt; (3) inhibits the expression and activation of key proteins responsible for the progression of cells into the DNA replicating phase (such as cyclin D1 expression and cdk4 activity associated with it and pRb phosphorylation); (4) upregulates the expression of the cdk inhibitor p27, suggesting that 2-ME can block HASMCs in G0/G1 phase via 2 different mechanisms; (5) inhibits cyclin B1 expression and cdk1 activity associated with it (essential for G2-to-M progression); (6) inhibits tubulin polymerization, a critical process in cell division; (7) induces COX-2 expression (known to mediate antiproliferative actions in SMCs); and (8) does not induce apoptosis, suggesting that the growth inhibitory effects of 2-ME are not attributable to apoptosis.
That 2-ME induces p27 suggests that 2-ME inhibits SMC proliferation via this negative regulator of cell growth. Indeed, downregulation of the cdk inhibitor p27 in response to mitogens is important for maximal activation of G1 cyclin/cdk complexes, and p27 rapidly falls in rat carotid arteries following balloon injury,10 allowing SMCs to proliferate in response to mitogenic signals. Therefore, upregulation of p27 expression by 2-ME may importantly contribute to the antiproliferative effects of 2-ME in SMCs.
In contrast to p27, 2-ME downregulates expression of the cdk inhibitor p21. Although p21 and p27 sequestration and ubiquitin-dependent degradation by proteasomes play a key role in their regulation and degradation, other ubiquitin-independent pathways for p21 degradation exist.11 Previous studies indicate that p21 expression is positively regulated by ERKs9 and that expression of p21, but not p27, is regulated by ERK1/2.12 Therefore, it is feasible that 2-ME attenuates p21 expression via the inhibitory action of 2-ME on ERK1/2 phosphorylation. Similar to 2-ME, PD98059 (an inhibitor of ERK1/2 phosphorylation) decreases p21 expression with no effect on Akt.
Pharmacological agents that block tubulin polymerization are potent inhibitors of SMC growth and prevent injury-induced neointimal thickening.13 Our finding that 2-ME inhibits tubulin polymerization and injury-induced intimal thickening suggests that 2-ME may be an effective therapeutic agent against vasoocclusive disorders. Similar to paclitaxel and taxol, 2-ME interferes with microtubule activity in cancer cells.5,14 Microtubules play an important role in governing the movement of chromosomes during mitosis, and this could possibly explain 2-ME-induced accumulation of HASMCs in G2/M phase. Although the exact receptors for 2-ME remain undefined, it is well established that 2-ME interacts with colchicine-binding sites on tubulin.5 Accordingly, we observed that colcemid, a colchicine analog, mimics the effects of 2-ME on tubulin polymerization in SMCs. Moreover, our previous studies show that similar to paclitaxel, 2-ME is a potent inhibitor of SMC migration and extracellular matrix formation.1
Apart from interference with tubulin, other mechanisms may participate in mediating 2-ME-induced arrest of HASMCs in G2/M phase. 2-ME inhibits the expression of cyclin B1 and cdk-1 activity associated with it, which is required for mitotic initiation.15 Indeed, inhibition of cyclin B1 expression by antisense prevents injury-induced neointima formation,10 and the majority of cancer cell lines overexpress cyclin B1,15 suggesting that the inhibitory actions of 2-ME on cyclin B1 may play a role in preventing neointima formation.
Our finding that 2-ME induces COX-2 expression in SMCs is consistent with the observations that tubulin-interfering agents stimulate COX-2 activity.5 Because prostacyclin inhibits SMCs growth,4,6 it is feasible that 2-ME inhibits SMC growth in part via this mechanism. Indeed, NS-398, a COX-2 blocker, shifted the inhibitory curve of 2-ME to the right. Recent findings indicate that the protective actions of estradiol against atherosclerosis are COX-2 mediated,6 and our previous findings demonstrate that the antimitogenic actions of estradiol are ER independent (not blocked by ICI182780) and attributable to its sequential conversion to methoxyestradiol.16 It is feasible, therefore, that COX-2 activity within the vascular bed is regulated via ER-dependent and -independent mechanisms by estradiol and 2-ME, respectively. This contention is supported by our observations that 2-ME inhibits neointima formation and, in obese rats, 2-ME reduces cholesterol and improves cardiovascular function.16
Estradiol upregulates endothelial NO synthase (NOS) via increased Akt phosphorylation,1 and, in intact vessels, endothelium-derived nitric oxide (EDNO) inhibits SMC growth.1 Our finding that 2-ME inhibits Akt phosphorylation in SMCs suggests that it may also inhibit Akt phosphorylation in endothelial cells and interfere with EDNO-associated antiproliferative actions. However, the findings that the inhibitory actions of estradiol are not lost in NOS-deficient mice1,16 and that 2-ME induces cardioprotective actions in a chronic model of NO inhibition17 suggests that mechanisms other than EDNO are active in mediating the antiproliferative actions. The above findings together with the fact that estradiol directly inhibits SMC growth and mitogen-activated protein kinase (MAPK) activity via sequential metabolism to methoxyestradiol1,16 suggests that 2-ME would prevent neointima formation via both endothelium-dependent and -independent mechanisms by targeting SMC growth, a prerequisite for neointima formation.
Compared with tubulin polymerization, the inhibitory effects of 2-ME on cyclins and signal transduction pathway proteins are observed at higher concentrations. Even though these pathways contribute to the regulation of SMC growth, it is unclear whether the observed effects of 2-ME are direct or simply “bystander” manifestations of a primary effect on the cytoskeleton. Lack of 2-ME-induced apoptosis, partial reversal of the inhibitory actions of 2-ME in the presence of COX-2 inhibitors, and downregulation of p21 expression suggest that interaction of 2-ME with pathways other than tubulin is in part responsible for the inhibitory effects of 2-ME on SMC growth. More detailed studies will be required to address this issue further. Also, the observation that the inhibitory effects of 2-ME on SMC proliferation were time dependent suggests that lower concentrations of 2-ME are active. Indeed, in in vivo experiments, the circulating levels of 2-ME are 3.274 ng/mL (10.8 nmol/L; see supplementary data) and are associated with inhibition of intimal growth and cell-proliferation. Similarly, in in vitro experiments, low concentrations (10 nmol/L) of 2-ME inhibit SMC proliferation after 4 to 8 days of treatment.
Our finding that 2-ME inhibits SMC proliferation by interfering with the cell cycle is consistent with observations by Gui and Zheng.18 Their finding that treatment of SMCs with 2-ME results in SMCs with 4N DNA is in agreement with our findings and may, in part, explain the accumulation of cells in G2/M phase. However, in contrast to mitotic-cell apoptosis observed by them, we do not see apoptosis in SMCs treated with 2-ME. We observe neither caspase-3 cleavage nor fragmentation of β-actin or accumulation of cells in sub-G1 population, all indirect indicators for apoptosis.19 Caspase-mediated cleavage of cytoskeletal β-actin plays a role in the morphological changes associated with apoptosis.19 We observe negligible numbers of apoptotic cells in carotid segments from 2-ME-treated rats, suggesting that the antivasoocclusive actions of 2-ME are attributable to its antiproliferative, rather than apoptotic, actions.
Although the reasons for the disparate findings remain unclear, differences in the methodology used and the experimental conditions (shorter treatment times, double FCS concentration) could be a potential factor. In this regard, Gui and Zheng observe apoptosis with no accumulation in G0/G1,18 whereas we observe accumulation in G0/G1 and no apoptosis as well as no loss of cells, reversibility of 2-ME inhibitory effects on treatment withdrawal, and no apoptosis in vivo. Moreover, when Gui and Zheng arrest cells in G1/S-phase, similar to our study, 2-ME-induced apoptosis is lost, suggesting that its apoptotic effects are cell-cycle driven. Additionally, the induction of p27 levels by 2-ME observed in our study explains the inhibitory effects of 2-ME on SMC proliferation and the accumulation of SMCs in G0/G1. In contrast, Gui and Zheng observe a downregulation of p27. The fact that those authors exclusively observe an arrest of cells in the mitotic phase suggests that the discrepancy may be a consequence of different experimental conditions.
Consistent with our findings, Seegers et al14 do not observe apoptosis in normal human skin fibroblasts treated with 2-ME. In cancer cells, p21 plays a key role in inducing the apoptotic effects of 2-ME.20 In fact, 2-ME-induced apoptosis is associated with sequential upregulation of p53 and p21 expression.20 The above findings, together with our observation that 2-ME decreases p21 expression in SMCs and carotid arteries, suggest that this major apoptotic pathway is not upregulated in vascular SMCs and may explain the lack of apoptotic effects in SMCs. Indeed, the status of p53-induced p21 expression plays a role in determining the apoptotic effects of 2-ME in various cell lines.14 Moreover, lack of or lower levels of p53 are associated with lack of apoptotic effects of 2-ME in various mammalian cells.20 The fact that 2-ME induces apoptosis in cancer cells or mitotic cells, but not in normal cells, may be of therapeutic importance for its potential use as an anticancer drug. However, further investigations are needed to evaluate this possibility.
Our findings have both therapeutic and pathophysiological relevance. The successful use of tubulin-interfering anticancer drugs like paclitaxel in preventing restenosis13 and our observation that 2-ME protects against injury-induced neointima formation suggest that 2-ME coated stents may protect against restenosis. Indeed, 2-ME inhibits key cell-cycle events in SMCs that contribute to vascular-proliferative diseases such as restenosis and bypass vein disease. Interestingly, 2-ME is a potent antiangiogenic agent that inhibits neovascularization and capillary formation,2 and may also be protective against plaque formation by inhibiting the growth of capillaries that nourish plaques. Because increased body weight is associated with cardiovascular disease and treatment with 2-ME reduces bodyweight, it is feasible that treatment with 2-ME may induce its cardioprotective actions in part via reduction of body weight. The facts that 2-ME is an endogenous metabolite with no affinity for ERs and that the antiproliferative actions of estradiol are in part mediated via its sequential conversion to methoxyestradiol1 suggest that estradiol metabolism within the coronary artery may be an important determinant of the cardiovascular protective actions of circulating estradiol.
In conclusion, 2-ME inhibits injury-induced neointima formation and blocks SMC proliferation by inhibiting key pathways involved in cell growth. Hence, 2-ME may be of therapeutic use against vasoocclusive disorders. The fact that 2-ME treatment does not alter testosterone levels or testis weight suggests that it could be of beneficial use in both men and women.
Limitations of Study
Although both the in vivo and in vitro studies provide strong evidence that 2-ME induces its antivasoocclusive actions by inhibiting SMC proliferation, it is difficult to draw a direct correlation between the dose/concentration effects observed in vitro and in vivo. Also, high concentrations (2 μmol/L) of 2-ME induced toxicity in leukemia cells.21 Although many of the effects in the present study were observed at comparable concentrations, our finding that the effects of 2-ME were reversible and that there was no cell loss suggest that the effects were not attributable to SMC toxicity. Indeed, even though the circulating levels of 2-ME in men and nonpregnant and pregnant women range between 10 to 35 pg/mL, 18 to 138 pg/mL, and 216 to 10 691 pg/mL, respectively, in a phase II clinical trial, no toxicity was observed in men given 400 and 1200 mg/d, with trough plasma levels of unconjugated 2-ME of 4 ng/mL. Nonetheless, further studies are required to assess its toxicity.3 Finally, the main mechanism by which 2-ME inhibits SMC proliferation remains to be further defined. Based on our data, it is unclear whether the modulatory effects of 2-ME on cell-cycle and signal-transduction pathways contribute to its inhibitory actions or whether these effects are simply bystander manifestations of a primary effect on the cytoskeleton.
Sources of Funding
Supported by grants from Swiss National Science Foundation (3200B0-106098/1), Oncosuisse (OCS-01551-08-2004), EMDO Stiftung, and the NIH (HL69846 and DK68575).
Original received January 26, 2006; revision received June 7, 2006; accepted June 12, 2006.
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