Effect Of Dicycloplatin On The Proliferation Of Human Aortic Smooth Muscle Cells And Human Aortic Endothelial Cells:in Vitro Study

BackgroundDrug-eluting stent (DES) has shown superior angiographic and clinical results compared with bare metal stent (BMS) in patient with coronary artery disease undergoing PCI. DES decreases late loss and restenosis by reduced neointimal proliferation following stenting, however it also inhibits re-endothelialization and vascular healing which can increase the incidence of in-stent thrombosis. We expect to approach a new drug which could inhibit the overgrowth of vascular smooth muscle cells (SMCs), and have no significant effect on re-endothelialization.Dicycloplatin is the third generation of platimum complex, developed by Xing Da Scientific Systems company, which own country has the independent intellectual property rights. At present the studies about dicycloplatin are in laboratory research and phrase Ⅲ clinical trial, which showed significant antitumor effects and safety characteristics, no studies about the effect on normal and benign proliferation cell and tissues is reported.ObjectiveThe objective of the study is to investigate the effects of dicycloplatin on proliferation of HASMC and HAEC in vitro and to approach the potential possibility of dicycloplatin to be used for DES. Human aortic smooth muscle cells (HASMC) and human aortic endothelial cells (HAEC) were cultured in vitro. In cytology level, MTS assay was used to evaluate the effect of dicycloplatin on the proliferation of HASMC and HAEC, flow cytometry was used to detect the effect of dicycloplatin on cell cycle. In protein molecular level, the proliferating cell nuclear antigen (PCNA), Bax, and Bcl-2protein expression were detected to evaluate the effects of dicycloplatin on proliferation and apoptosis in HASMC and HAEC. Methods1. MTS assay to evaluate the effects of dicycloplatin on proliferation of HASMC and HAEC.HASMC and HAEC were cultured in vitro. HASMC and HAEC were incubated with different concentration of dicycloplatin for24hours,48hours, and72hours, cell proliferation was measured by MTS assay to explore if the efficacy is time and dose dependent.Double diluted dicycloplatin from100μg/ml to0.39μg/ml was incubated with HASMC for72hours; double diluted dicycloplatin from200μg/ml to1.56μg/ml was incubated with HAEC for72hours, cell proliferation was measured by MTS assay, log(concentration) and growth rate were used to explore the50%inhibited concentration(IC50) for HASMC and HAEC.HASMC and HAEC were incubated with different concentration of dicycloplatin (10μg/ml,1μg/ml,100ng/ml,10ng/ml,1ng/ml) for72hour, rapamycin as positive control groups, cell proliferation was measured by MTS assay, SPSS software, One-Way ANOVA method were used to analyze the minimum effective concentration.2. Flow Cytometry to detect the influence of dicycloplatin and rapamycin on the HASMC and HAEC cell cycleHASMC and HAEC were cultured for24hours, then cultured with serum-free smooth muscle cell medium (SMCM) or endothelial cell medium (ECM) for24hours. HASMC were divided into6groups:negative control group, lng/ml of rapamycin group;1μg/ml,100ng/ml,10ng/ml,1ng/ml dicycloplatin groups. HAEC were divided into6groups:negative control groups,10ng/ml of rapamycin group,10μg/ml,1μg/ml,100ng/ml,10ng/ml of dicycloplatin groups. Cell samples were collected48hours after incubation, the samples were fixed in low temperature, PI dyeing for30min, the cell cycle was detected by flow cytometry.3. Western Blot to detect the effect of dicycloplatin on PCNA, Bax and Bcl-2protein expression in HASMC and HAEC.The divided group, treatment with different concentration of dicycloplatin and rapamycin were same as above. Protein samples were collected48hours after incubation. Protein expression of PCNA, Bax, Bcl-2were analyzed using Western blot.Result1. The effects of dicyclopaltin on proliferation of HASMC and HAEC.For HASMC incubated with lOμg/ml dicycloplatin for24hours,48hours,72hours, the proliferation inhibiting rate were16%,39%and75%, respectively; the inhibiting rate in1μg/ml group were13%,13%and27%, respectively; the inhibiting rate in100ng/ml group were11%,9%and20%. For HAEC incubated with lOμg/ml dicycloplatin for24hours,48hours,72hours,the proliferation inhibiting rate was6.41%,11.05%and14.83%, respectively; the inhibiting rate in1μg/ml group were1.35%,0.60%and1.3%, respectively. The inhibiting efficacy of dicycloplatin is in a dose and time dependent manner.The drug concentration at which maximal HASMC and HAEC proliferation were inhibited by50%(IC50) were3.47μg/ml and72.44μg/ml respectively. The IC50for HAEC is20.88times higher than that for HASMC.Dicycloplatin in lOng/ml-lOμg/ml remarkably inhibited proliferation of HASMC (P<0.05), compared with the negative control group. Dicycloplatin in lng/ml had no proliferation inhibiting effect on HASMC (P=0.83). Dicycloplatin in10μg/ml remarkably inhibited proliferation of HAEC (P<0.05), dicycloplatin in1ng/ml-1μg/ml had no proliferation inhibiting effect (P>0.05). The minimum effective inhibiting concentration for HAEC was103-times higher than it for HASMC. The positive control group, rapamycin in lng/ml inhibited proliferation both for HASMC and for HAEC, and the effect was weaker in HAEC than in HASMC.2. Cell cycle analysis demonstrated that:for HASMC, dicycloplatin in1μg/ml arrested cell cycle in G2/M phase; in lOng/ml-100ng/ml arrested in S phase. For HAEC, dicycloplatin in10μg/ml arrested cell cycle in S phase; the rest groups showed no significant difference in cell cycle. Dicycloplatin was a non-specific inhibiter on cell cycle. In all groups, there were no apoptosis peak (Sub-G1) detected.3. In HASMC, PCNA expression remarkably decreased in all dicycloplatin groups compared to negative control group, Bax expression was increased in all dicyclopaltin groups, Bcl-2expression was increased in dicyclopatin1μg/mL group, decreased in10ng/ml and lng/ml groups. The ratio of Bax/Bcl-2was increased in all dicycloplatin groups. In HAEC, PCNA expression was normal or decreased slightly compared with negative control group, Bax expression was increased in all dicycloplatin groups. In the same experiment condition, Bcl-2was not detected in HAEC.ConclusionThe inhibiting effect of dicycloplatin on proliferation of HASMC is in a dose and time dependent manner. The IC50for inhibiting HAEC proliferation is about20.88times higher than that for HASMC. The minimum effective concentration of dicycloplatin for inhibiting proliferation of HAEC is103-times higher than that for HASMC.Cell cycle analyze show that dicycloplatin is a non-specific inhibiter on cell cycle.At the same concentration, dicycloplatin inhibits PCNA expression more remarkably in HASMC than in HAEC. In the mitochondrion apoptosis pathway, dicycloplatin regulates the expression of Bax and Bcl-2, even in low concentratin, dicycloplatin can promote apoptosis in HAEC and HASMC. This effect is only detected in protein level, in cell cycle analysis there are no apoptosis peak detected.In summery, although in Western blot experiment apoptosis associate protein expression is up regulated, there are no apoptosis peak(Sub-G1) detected in cell cycle analysis. In low concentration, dicyclopatin inhibits cell proliferation rather than promotes apoptosis. Similar with rapamycin, dicyclopatin significantly inhibits proliferation of HASMC in nanogram level, but the effect is weaker than rapamycin. Different to rapamycin, dicycloplatin inhibiting proliferation of HAEC needs much higher concentration. This characteristic of dicycloplatin may have potential to be used for a drug carried by DES.