VSMC proliferation plays an important role in the development of atherosclerosis lesions [17
], and, hence, the inhibition of VSMC proliferation might be a chief beneficial approach for atherosclerosis-related diseases [18
]. In the present study, we identified ketamine, a noncompetitive antagonist of the NMDA receptor, as a potent antiproliferative agent for VSMCs. A literature hunt indicated that ketamine has analgesic and amnesic effects, and has potential anti-depressant properties [19
]. A number of studies have found that high-dose ketamine treatment induces aberrantly high levels of neuroapoptosis in rodents and nonhuman primates [21
]. To date, there is no study for the effect of ketamine on cellular proliferation. Therefore, for the first time, this study examined the effect of ketamine on rat vascular smooth muscle cell (VSMC) proliferation. Our data clearly demonstrated that ketamine significantly inhibited PDGF-BB-induced VSMC proliferation via suppression of the cell survival signaling pathways, such as PI3K, Akt, and ERK. This study also demonstrated that PP2A plays a major role in ketamine’s inhibitory effects on VSMC proliferation. This study concludes that ketamine may be a potential candidate for the prevention and treatment of vascular inflammatory diseases.
Among the mitogen-activated protein kinase (MAPK) family, ERK1/2 has been majorly involved in the growth of various cell types [22
]. Akt, a serine/threonine protein kinase found to be activated via the PI3K pathway, has been involved in VSMC proliferation, cell cycle progression, and cell survival [23
]. The current study shows that ketamine is able to suppress the increased proliferation in the presence of PDGF-BB, and this inhibitory effect was boosted by combined treatment with PI3K inhibitor LY294002 and ERK inhibitor PD98059. These results indicate that the Akt and ERK pathway may be involved in the ketamine-mediated inhibition of VSMC proliferation. PI3K/Akt is recognized as one of the major signaling molecules for cell proliferation and survival facilitated by extracellular stimuli [24
]. Substances for suppression of the PI3K/Akt pathway have used extensively in the treatment of hypertension and angina, and display a range of biological properties in the cardiovascular system. A previous study found that PI3K/Akt is greatly expressed in human as well as murine atherosclerotic lesions, and PI3K inhibitor AS605240 considerably reduced these lesions in apolipoprotein E (Apo-E
)-null mice [25
]. These authors have also found that this PI3K inhibitor was effective on advanced atherosclerotic lesions of low-density lipoprotein (LDL)-receptor-deficient mice, and suggested that PI3K might be a promising target for the treatment of atherosclerosis [25
]. Moreover, topotecan, a water-soluble camptothecin analog, is reported to downregulate the PI3K/Akt signaling pathway for the inhibition of vascular endothelial growth factor (VEGF)- and basic fibroblast growth factor (bEGF)-induced vascular endothelial cell migration [26
]. In some essential biologic processes, the PI3K/Akt inhibitors have been used extensively as pharmaceutical tools and accompanying signaling pathways [27
]. Hence, this perception into the role of the PI3K/Akt in human diseases may offer a wide spectrum of therapeutic strategies. In harmony with the above evidence, this study found that increased PI3K, Akt, and ERK1/2 phosphorylation stimulated by PDGF-BB was potently inhibited by ketamine. These results indicate that inhibiting PDGF-BB-induced activation of the PI3K, Akt, and ERK1/2 signaling pathway may have contributed to the inhibition of VSMC proliferation exerted by ketamine.
PP2A is one of the major Ser/Thr phosphatases associated with the regulation of various cellular processes [28
]. PP2A controls numerous cell-signaling pathways by triggering dephosphorylation of various signaling proteins. A previous study has shown that PP2A can directly dephosphorylate Akt and ERK [29
]. A report indicated that treatment with PP2A inhibitor okadaic acid (OA) not only inhibited PP2A activation, but also triggered Akt and ERK signaling and resulted in increases in cell growth, migration, and angiogenic ability, consequently endorsing the anti-proliferating effect of PP2A in endothelial cells [30
]. An in vitro cell model study also found reduced PP2A activity and high levels of AKT and ERK phosphorylation in both primary TG (+) HEC cells and human HEC-P cells. This evidence supports our finding that ketamine-inhibited PDGF-BB-induced PI3K/Akt/ERK phosphorylation was significantly restored by both PP2A inhibitor okadaic acid and pp2a siRNA. Together, these results show that PP2A regulation plays a major role in ketamine’s inhibitory effect on VSMC proliferation.
PP2A activity is regulated by several molecular events, such as post-translation modification, auto-regulation, and substrate protein interaction. PP2A catalytic efficiency is reported to be controlled by two major alterations: phosphorylation [31
] and methylation [32
]. Tyrosine (Tyr) and leucine (Leu) undergo phosphorylation and methylation, respectively, and phosphorylation of Tyr307 successfully causes a decline in PP2A activity by preventing the interaction with PP2A-C [33
]. Autophosphorylation-activated protein kinase is reported to inactivate the protein tyrosine phosphatase activity of PP2A [34
]. PP2A-C expression is firmly controlled in the cell at the translational level, but not at the transcription level [35
]. PP2A’s reversible methylation is a preserved controlling mechanism, and it has been reported that the methylation of Leu309 in a preserved TPDYFL motif in the C terminus of PP2A-c enhances holoenzyme assembly and phosphatase activity [36
]. In addition to reversible methylation, PP2A-c has also been shown to endure tyrosine phosphorylation at Y307. Nevertheless, in contrast to methylation, tyrosine phosphorylation has been established to obstruct the catalytic function of PP2A [37
]. In the present study, pp2a
siRNA alone or combined with ketamine significantly inhibited the PDGF-BB-induced PP2A-C expression. Moreover, this study also found ketamine significantly and concentration-dependently suppressed the phosphorylation and demethylation of PP2A in VSMCs. These notable results further support our finding that ketamine inhibits PDGF-BB stimulated proliferation in VSMCs by the regulation of PP2A.
Our previous study had demonstrated that various concentrations of ketamine (100–500 μM) used to treated microglia did not cause cytotoxicity, and, at the higher concentration (500 μM), significantly inhibited TNF-α production [38
]. Shibakawa et al. reported that ketamine at 100, 300, or 1000 µM significantly inhibited some of the inflammatory responses in microglial cells stimulated by lipopolysaccharide (LPS) without inducing cytotoxicity [39
]. Moreover, it was reported that ketamine was used at up to 160 mg, which is equivalent to approximately 650 µM, in patients who had undergone retroperitoneal node dissection hemiscrotectomy surgery [40
]. Therefore, we used this study a non-cytotoxic high concentration of 500 µM ketamine for its antiproliferative mechanistic effects in VSMCs.