Percutaneous coronary intervention has become a prominent treatment modality for coronary artery disease. However, restenosis has limited the long-term success of balloon angioplasty and bare metal stents. The key mechanism of restenosis after bare metal stents implantation is proliferation and migration of vascular smooth muscle cells;1 although vascular smooth muscle cells are in the quiescent phase of the cell cycle or in a low proliferating state, they can be stimulated to proliferate during the pathological processes.2,3 The restenosis-prevention effects of sirolimus and paclitaxel, which are being successfully used in drug-eluting stents (DES), are based on apoptosis induction and proliferation inhibitions.4–6 The development of DES to combat the problem of in-stent restenosis has revolutionized interventional cardiology. DES dramatically decreased in both angiographic restenosis and the need for target lesion revascularization over prior bare metal stens counterparts.7–9 However, concerns have emerged about the risk of late stent thromboses associated with DES.10,11
The characteristics of the ideal anti-restenotic drug were defined by Sousa et al.12 The ideal agent should exert anti-proliferative effects without inhibiting vascular healing, have wide therapeutic-to-toxic ratio, and not induce inflammation or thrombosis. The physicochemical properties of the compound should be balanced between hydrophobicity and hydrophilicity. Obviously, current drugs used in DES do not meet the aforementioned ideal agent characteristics and the search for the ideal anti-restenotic agent continues.
Platinic compounds, which are mental-coordination antineoplastic agents, destroy DNA structure, function and replication. Dicycloplatin is a third generation platinic compound developed by Beijing Suo Pu Xing Da Pharmaceutical Co. Ltd., China. Among distinctive physicochemical properties of the dicycloplatin molecule relative to carboplatin and cisplatin are its hydrophilicity, stability in acid medium, and a lipid/water partition coefficient ClogP of 1.0.13 The DNA disruption action of dicycloplatin was reported similar to that of carboplatin and cisplatin. Laboratory and phase III clinical studies of dicycloplatin have documented safety and significant anti-tumor effect. Because of its safety we posited that dicycloplatin might be useful at low doses for inhibiting benign proliferation, such as that of vascular smooth muscle cells after stenting. No studies on effects of the drug on normal and benign proliferation have been reported.
The present study was designed to assess the effects of dicycloplatin on in vitro proliferation of human aortic smooth muscle cells (HASMC) and human aortic endothelial cells (HAEC) as groundwork to establish its potential for use in DES.
HASMC and HAEC as cryopreserved cultures, and smooth muscle cell medium and endothelia cell medium with growth supplements were purchased from ScienCell Research Laboratories (San Diego, CA, USA); dicycloplatin from Beijing Suo Pu Xing Da Pharmaceutical Co. Ltd., CellTiter 96 AQueous One Solution Cell Proliferation kit from Promega (WI, USA), and PI and RNase from Sigma-Aldrich (MO, USA). The spectrophotometer was manufactured by Tecan Company (Männedorf, Switzerland) and the flow Cytometer by Beckman Coulter Company (CA, USA)
HASMC were cultured in smooth muscle cell medium with growth supplements and fetal bovine serum (2% vol/vol), while HAEC were cultured in endothelial cell medium with growth supplements and fetal bovine serum (5% vol/vol). Cells were cultured in a humidified atmosphere of 5% CO2 and 95% air. Cells from passages 4–6 were used in the experiments.
Cell proliferation assay
Time- and concentration-dependent detection
HASMC were seeded at a density of 1000 cells/well in 96-well plates; after 24 hours, the medium was replaced with fresh one containing dicycloplatin at concentrations of 100 ng/ml, 1 μg/ml and 10 μg/ml; while HAEC were seeded at a density of 2000 cells/well in 96-well plates; after 24 hours, medium was replaced with fresh one containing dicycloplatin at concentrations of 1 μg/ml and 10 μg/ml. Cells were then incubated with dicycloplatin for additional 24, 48 and 72 hours, after which (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxypheny l)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) solution was added to each well to a final concentration of 317 μg/ml, and incubated for 3 hours to allow MTS reduction. Absorbance was measured at 490 nm with the spectrophotometer. Cell inhibition rate was calculated as = (1 - ODtreatment / ODcontrol) × 100%. Each experiment was performed 5 times.
HASMC and HAEC were seeded in 96-well plates and incubated for 24 hours, then the medium was replaced with fresh one containing dicycloplatin at various concentrations (double dilution) ranging from 0.39 μg/ml to 100 μg/ml for HASMC or between 0.78 μg/ml and 200 μg/ml for HAEC. Cells were then incubated with dicycloplatin for an additional 72 hours. OD values were then measured; IC50 values correspond to the dicycloplatin concentration that reduced mean absorbance at 490 nm to 50% of that in untreated control cells.
Minimal effective concentration detection
HASMC and HAEC were seeded in 96-well plates and incubated for 24 hours, then the medium was replaced with fresh one containing dicycloplatin at concentrations ranging from 1 ng/ml to 10 μg/ml. Sirolimus was added to a final concentration of 1–100 ng/ml as a positive control. Cells were then incubated with dicycloplatin or sirolimus for an additional 72 hours. After the incubation period, OD values were measured, and cell inhibition rate was calculated.
Flow cytometic cell cycle analysis
HASMC and HAEC were cultured in 6-well plates, starved by serum deprivation for 24 hours, then the quiescent HASMC were left resting and stimulated with dicycloplatin at concentrations of 1 ng/ml-1 μg/ml or sirolimus at a concentration of 1 ng/ml. HAEC were left resting and stimulated with dicycloplatin at concentrations of 10 ng/ml-10 μg/ml or sirolimus at a concentration of 10 ng/ml. After 48 hours, HASMC and HAEC were harvested by trypsinization, and after fixation in ice-cold 75% ethanol, cells were washed and resuspended in phosphate buffered saline (PBS) containing 25 μg/ml RNase and 0.5% Triton X-100. Samples were then incubated with 50 μg/ml propidium iodide for 30 minutes and analyzed by flow cytometry.
Western blotting assay
HASMC were incubated with dicycloplatin at concentrations of 1 ng/ml-1 μg/ml, and sirolimus as a positive control was added at a concentration of 1 ng/ml. HAEC were incubated with dicycloplatin at concentrations of 10 ng/ml-10 μg/ml, and sirolimus was added at a concentration of 10 ng/ml. Untreated cells were taken as negative control groups.
After 48 hours, cells were rinsed twice with ice-cold PBS and lysed in 100 μl/well lysis buffer. Lysates were disrupted further by running several times, then clarified by centrifugation at 14 000 r/min for 15 minutes. The supernatant fluid was collected and mixed with loading buffer. Total protein was resolved by 12% sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane. The transfer membrane condition was 25 V, 25 minutes for p53; 18 V, 15 minutes for proliferating cell nuclear antigen (PCNA) and Bax. The membrane was blocked for 1 hour at room temperature in PBS containing 0.05% Tween 20 and 3% milk powder, followed by 2-hour incubation with primary antibody; the primary antibodies used were anti-PCNA (1:1000), anti-Bax (1:200), anti-p53 (1:200) (all from Cell Signaling Technology, Beverly, MA, U.S.A); anti-glyceraldehyde phosphate dehydrogenase (GAPDH) (1:2000). The membrane was washed several times with blocking solution and incubated for an additional hour with secondary antibody (1:1000). After washing, proteins were detected using an enhanced ECL reagent (Thermo, U.S.A). Optical density of protein bands was analyzed with Quantity One software (Bio-Rad, U.S.A).
Experiments results are expressed as mean ± standard deviation (SD). Two-way analysis of variance (ANOVA) was used for analysis of time-and concentration -dependent experiment results; one-way ANOVA was used for analysis of the rest of experiment results. A P value of less than 0.05 for the ANOVA was considered statistically significant. SPSS 10.0 (SPSS Inc., USA) was used for data analysis.
Dicycloplatin inhibits cell proliferation in HASMC and HAEC
Time- and concentration-dependent detection
HASMC and HAEC were incubated with different concentrations of dicycloplatin for 24, 48 and 72 hours, and proliferation was measured by MTS assay. Two-way ANOVA analysis proved that inhibition was time- and concentration-dependent (Figure 1).
Based on the MTS assay, IC50 was 3.47 μg/ml and 72.44 μg/ml for dicycloplatin treated HASMC and HAEC, respectively (Figure 2).
Minimal effective concentration detection
The effects of dicycloplatin on HASMC proliferation were measured with MTS assay. Figure 3A shows the antiproliferative effects of dicycloplatin on HASMC after 72-hour treatment. Cell proliferative rate decreased 75% at the highest dicycloplatin concentration (10 μg/ml) compared with that in the negative control. Antiproliferative effects of dicycloplatin remained significant even at concentrations as low as 10 ng/ml. For comparison, the antiproliferative effects of sirolimus was significantly stronger than that of dicycloplatin, and sirolimus at 1 ng/ml yielded significant antiproliferative effects on HASMC (P <0.05) (Figure 3B). The effects of dicycloplatin on proliferation of HAEC after 72-hour treatment are shown in Figure 3C. Cell proliferation rate decreased only by 14.8% at the highest dicycloplatin concentration (10 μg/ml) compared with that of the negative control. There were no significant antiproliferative effects on HAEC at dicycloplatin concentrations from 1 ng/ml to 1 μg/ml. In contrast, the anti-proliferative effect of sirolimus on HAEC was basically similar to that on HASMC (Figure 3D).
Flow cytometric cell cycle analysis
To characterize the contribution of cell cycle arrest to the reduction in HASMC and HAEC proliferation, flow cytometric analysis was performed for DNA content (Figure 4). HASMC cell cycle was arrested at the G2/M phase at a dicycloplatin concentration of 1 μg/ml (P <0.01), however the cell cycle was arrested at the S phase at concentrations of 10–100 ng/ml (P <0.01). In contrast, the HAEC cell cycle was arrested at the S phase at a high dicycloplatin concentration of 10 μg/ml (P <0.01), while no dicycloplatin effects on cell cycle were apparent at concentrations below 10 μg/ml. Sirolimus arrested the cell cycle at the G0/G1 phase in both HASMC (P=0.015) and HAEC (P <0.01). These data suggest that inhibition of HASMC and HAEC proliferation by dicycloplatin might be nonspecific.
Western blotting analysis
PCNA expression in HASMC remarkably decreased in all dicycloplatin concentration groups compared to the negative control group (P <0.01), and similar to that in the sirolimus group. Bax expression was increased in all dicycloplatin groups (P <0.01) while there was no significant change in the sirolimus group. The p53 expression was increased in the sirolimus and dicycloplatin treated groups at concentrations of 100 ng/ml and 1 μg/ml, respectively. The results are shown in Figure 5.
PCNA expression in HAEC was significantly decreased in the sirolimus group (P <0.01), while there were no significant changes for all dicycloplatin groups at concentrations ranging from 10 μg/ml to 10 ng/ml relative to the negative control group. Bax expression was increased in all dicycloplatin groups (P <0.01), but slightly decreased in the sirolimus group. The p53 expression was decreased in cells treated with dicycloplatin at a high concentration of 10 μg/ml, but it was increased at concentrations of 1 μg/ml and 100 ng/ml. The p53 expression was slightly increased in the sirolimus groups, but without statistically significant difference (Figure 6).
A meta-analysis showed that the very late stent thrombosis rate of first-generation DES was slightly but significantly higher than that of bare metal stents.14 Late stent thrombosis was partly attributed to delayed endothelial healing after DES implantation. Except for strut fractures, stent malapposition, and plaque rupture, among others, the biologic effects of the drug and polymer coating are probably the most important causes of late thrombosis, as they are unique to DES. The durable polymer in DES induces inflammation and hypersensitivity reactions which may delay endothelial healing.15 The drugs carried by DES significantly inhibit vessel smooth muscle cell proliferation, which leads to decreased restenosis risk, but also inhibits endothelial cell proliferation, which may underlie stent thrombosis. The present study revealed that sirolimus has potent anti-proliferative effects on both HASMC and HAEC at almost similar concentration levels. Marx et al16 reported that sirolimus at concentrations as low as 1 ng/ml inhibited DNA synthesis and cell growth in vascular smooth muscle cells. Liu et al17 revealed that sirolimus above the concentration of 10 ng/ml had antiproliferative effects on HAEC. Results of this study resemble those previously reported with sirolimus having antiproliferative effects on both HASMC and HAEC at the nanogram level, which might explain why sirolimus inhibits proliferation of smooth muscle cells but at same time delays endothelial healing.
HASMC and HAEC viability 72 hours after dicycloplatin treatment was quantified using the MTS assay based on reduction of tetrazolium salt to colored formazan by dehydrogenase enzyme activity that only exists in mitochondria of viable cells. In the present study, we found that dicycloplatin had antiproliferative effects on HASMC and HAEC, and the effects were concentration and time dependent. At dicycloplatin concentrations of 10 ng/ml or higher, HASMC proliferation inhibition rate increased with drug concentration. However, dicycloplatin had antiproliferative effects on HAEC only at concentrations above 10 μg/ml. Antiproliferative effect of dicycloplatin on HAEC was significantly weaker than that on HASMC; the concentration of dicyloplatin needed for a significant antiproliferative effect on HAEC is around 1000 times higher than that on HASMC. The present study also revealed that IC50 was 3.47 μg/ml and 72.44 μg/ml in dicycloplatin treated HASMC and HAEC, respectively. This unique feature of dicycloplatin, i.e., significantly inhibiting smooth muscle cell proliferation while only gently inhibiting endothelial cell proliferation suggests a potential use of dicycloplatin as the drug component of DES to minimize occurrence of both restenosis and stent thrombosis.
Cell cycle analysis showed that dicycloplatin was a non-specific cell cycle inhibitor. In the present study, various dicycloplatin concentrations arrested cell cycle at different phases. At the same concentration, dicycloplatin arrested the cell cycle at different phases in HASMC and HAEC. It has been reported18 that platinic compounds' arrest of the cell cycle is dependent on cell type, a differential characteristic with sirolimus. Sirolimus arrests the cell cycle at the G0/G1 phase as also shown in the present study and in previous reports, 16,19 and it binds to the cytosolic protein known as the FK506 binding protein (FKBP12), inhibits cell-cycle progression via upregulation of the cyclin-dependent kinase inhibitor, p27kip1. Dicycloplatin binds to DNA and destories DNA structure, inhibited cell cycle non-specific, indicating that dicycloplatin is cytotoxic, similiar to paclitaxel. But in this study, dicycloplatin exhibited minor antiproliferation effect on HAEC. This characteristic could make dicycloplatin be a drug carried by DES better than paclitaxel.
After stents are delivered to the coronary artery, injury to endothelial cells and exposure of collagen promote platelet adherence and aggregation with release of mitosis growth factors and cytokines. These factors promote the proliferation and migration of vascular smooth muscle cells through cell signal conducting pathways, which Speir et al20 have suggested concentrate in the G1-S phase and lead to DNA replication, thus cells enter and complete the S phase.21 PCNA is the essential protein that stimulates cells into entering the cell cycle and initiating DNA replication. The identification of PCNA as a processivity factor for replicative DNA polymerases placed it at the heart of the replisome. Pol-α primase synthesizes the first RNA/DNA primer on the leading strand, then pol δ, together with its processivity factor PCNA, performs continuous leading strand synthesis. 22 In the present study, PCNA expression in HASMC remarkably decreased in all dicycloplatin groups as compared to the negative control group, while PCNA expression in HAEC was not significantly changed. These results are consistent with those of the MTS assay, which indicates that dicycloplatin inhibits proliferation of HASMC and HAEC at both cell and protein levels. The effect of sirolimus on PCNA expression in HASMC is similar to that of dicycloplatin, however, the effect of sirolimus on PCNA expression in HAEC is significantly greater than that of dicycloplatin.
Apoptosis is a form of genetically programmed cell death, which plays a key role in regulation of cellularity in a variety of tissues and cell types. Bcl-2 family and p53 are common mitochondria apoptosis related proteins. Bcl-2 family proteins form a complex regulatory network that controls cell survival and death in response to different physiological and pathological stimuli.23 Many cytotoxic substances may attack mitochondria, translocate pro-apoptotic members of the Bcl-2 family to mitochondria, alter voltage-dependent channels in the mitochondrial membrane, and release cytochrome-c (cyt-c). The Bax protein is a member of the Bcl-2 family that promotes apoptosis.24 Nuclear proteins, in particular those related to oncogenes or anti-oncogenes,25 such as p53, also play important roles in the regulation of apoptosis. The tumor suppressive gene p53 functions as an anti-oncogene associated with up-regulation of apoptosis; wild-type p53 arrests cell proliferation and may hold cells with DNA damage in the G1 phase. During DNA repair, some p53 expressing cells oppose the G1 block and enter the suicide pathway. Autophagy is regulated by an intricate network of signaling cascades that have not yet been entirely disentangled. Accumulating evidence indicates that p53 can modulate autophagy in a dual fashion, depending on its subcellular localization.26 On the one hand, p53 functions as a nuclear transcription factor and transactivates proapoptotic, cell cycle-arresting and proautophagic genes. On the other hand, cytoplasmic p53 can operate at mitochondria to promote cell death and can repress autophagy via poorly characterized mechanisms. In this study, we detected Bax and p53 protein expression by Western blotting analysis. After HASMC and HAEC treatment with dicycloplatin, the mitochondria apoptosis pathway was active, Bax protein expression in HASMC and HAEC increased in all dicycloplatin groups, but not in a concentration-related manner; p53 protein expression in HASMC was upregulated in the high concentration groups, in apparently a concentration-dependent manner. The p53 expression in HAEC was reduced at a high dicycloplatin concentration of 10 μg/ml, while it was increased at concentrations of 100 ng/ml-1 μg/ml. This phenomenon suggests that apoptosis may play a certain role in the antiproliferative effect of dicycloplatin, but may not be a major mechanism.
In conclusion, although the effect is relatively weaker than that of sirolimus, dicycloplatin at nanogram levels significantly inhibits HASMC proliferation and at much higher doses and lesser extent HAEC proliferation. The minimum effective concentration of dicycloplatin necessary to inhibit proliferation of HAEC is 1000 times higher than that of HASMC. At the same concentration, dicycloplatin inhibits PCNA expression more significantly in HASMC than in HAEC. Cell cycle analysis shows that dicycloplatin is a non-specific cell cycle inhibitor. In the mitochondrial apoptosis pathway, dicycloplatin upregulates the expression of Bax and p53 and promotes apoptosis in HAEC and HASMC at the molecular protein level to some degrees. The latter effects of dicycloplatin suggest that it potentially might be used in DES.
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