Effect of S-diclofenac, a novel hydrogen sulfide releasing derivative inhibit rat vascular smooth muscle cell proliferation

https://doi.org/10.1016/j.ejphar.2008.07.029Get rights and content

Abstract

S-diclofenac (2-[(2,6-dichlorophenyl) amino] benzene acetic acid 4-(3H-1,2,dithiol-3-thione-5-yl) phenyl ester) is a novel molecule comprising a hydrogen sulfide (H2S)-releasing dithiol-thione moiety attached by an ester linkage to diclofenac. Effect of S-diclofenac (H2S donor) on cell proliferation was investigated on the primary and immortalized rat aortic vascular smooth muscle cells (SMC). Smooth muscle cell proliferation has been considered as a key event in vascular injury in diseases such as atherosclerosis and restenosis after invasive intervention. Clonogenic cell survival assay showed a dose dependent (10–100 μM) decrease in cell survival. Flow cytometric analysis showed that the asynchronized cells are more sensitive than the cells that are synchronized and revealed that the cells in G1 phase are not affected by the treatment of the S-diclofenac. Asynchronized smooth muscle cells treated with the S-diclofenac showed an increase in apoptotic cell death. S-diclofenac treatment also resulted in stabilization of p53 coupled with the induction of downstream proteins such as p21, p53AIP1 and Bax. S-diclofenac did not up-regulate cell levels of the antiapoptotic protein Bcl-2. However, when the cells are synchronized a stimulatory effect of cell growth with the decrease in apoptosis, p53 and p21 was evident. S-diclofenac inhibits smooth muscle cell growth and may play a role in the lesion formation at sites of the vascular injury. The present results suggest that S-diclofenac may be useful for the prevention of smooth muscle cell proliferation in diseases such as vascular obstructive and restenosis.

Introduction

Hydrogen sulfide (H2S) is produced in nature primarily through the decomposition of dead plant and animal matter by anaerobic sulfur bacteria (Beauchamp et al., 1984). It is now becoming increasingly apparent that H2S is also synthesized naturally from l-cysteine in mammalian tissues in a reaction catalyzed by two enzymes, cystathionine-γ-lyase and cystathionine-ß-synthetase (Wang, 2002, Kamoun, 2004). Thus, whilst H2S was once considered as an environment pollutant, has transited rapidly to biologically relevant mediator with potential roles in several physiological processes and disease states in the body (Wang, 2002, Szabo, 2007). It has been shown, for example, that H2S plays a role in the regulation of vascular function in both normal and disease state (Zhao and Wang, 2002, Yusuf et al., 2006). Vascular smooth muscle cell proliferation has long been considered to be a key event in the remodeling of vascular wall following vascular injury in diseases such as atherosclerosis and vascular restenosis after invasive intervention (Dzau et al., 2002). More recently, the precise role of cell proliferation in atherogenesis has received more attention including muscle contraction, cell migration and apoptosis (Gizard et al., 2005).

The recent discovery of H2S as an endogenously produced gaseous second messenger capable of modulating many physiological process in the body including vasodilation (Wang, 2002, Wang, 2003), like that of nitric oxide (NO) an another gasotransmitter, prompted us to investigate the potential role of H2S on the smooth muscle cell growth. Recently, it has been reported that H2S exhibits vasodilation (Yusuf et al., 2006), mediates the vasoactivity of garlic (Benavides et al., 2007) and attenuates myocardial ischemia–reperfusion injury by preservation of mitochondrial function (Elrod et al., 2007) in rats. Therefore, the novel gasotransmitter H2S is emerging apart from nitric oxide is an important physiological mediator in the cardiovascular system. Cell growth is controlled by multiple, interrelated signaling pathways with many cell cycle regulatory checkpoints involved. However, the precise mechanism(s) by which H2S alters cell growth and its role in controlling the cell cycle is not well understood (Baskar et al., 2007a), but the recent works suggests that the precise role of H2S on the cardiovascular function is likely to be more complex (Szabo, 2007). In this study, we have examined the effects of a novel H2S donor; S-diclofenac (Isenberg et al., 2007, Li et al., 2007, Sidhapuriwala et al., 2007, Wallace et al., 2007, Rossoni et al., 2008) on cell survival, cell cycle alterations and proteins associated with cell growth and apoptosis in rat aortic vascular smooth muscle cells.

Section snippets

Cell culture and treatment

Normal rat aortic (A-10) and immortalized with SV40 (CRL-2018) smooth muscle cells (SMC) were obtained from the American Type Culture Collection (Manassas, VA, U.S.A). Cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% v/v fetal bovine serum (FBS), 1% of l-glutamine, 100-units/ml penicillin and 100-μg/ml streptomycin. All reagents were obtained from GIBGO, Carlsbad, CA, U.S.A. The cultures were maintained at 37 °C in a humidified 5% CO2 atmosphere. A novel H2

Cytotoxicity by cell survival assay

In the present study, we have studied the effects of S-diclofenac (a novel H2S donor) and its parent molecule diclofenac on the cell proliferation of rat aortic vascular smooth muscle cells. In a preliminary study, we first investigated the effect of S-diclofenac (10–100 μM) on cell survival by the colony forming efficiency on the primary (A-10) and immortalized with SV40 transformed smooth muscle cells. DMSO was used as a vehicle to dissolve the diclofenac and S-diclofenac. We have conducted

Discussion

In recent years, it has become clear that hydrogen sulfide (H2S) plays a number of biological roles (Szabo, 2007) and functions as a novel gasotransmitter in the body alongside nitric oxide (NO) and carbon monoxide (CO) (Wang, 2002, Wang, 2003, Moore et al., 2003). Recently, it has been reported that H2S acts as a vasodilation agent in rats (Zhao et al., 2001, Yusuf et al., 2006) and also mediates the activity of garlic (Benavides et al., 2007) and attenuates myocardial ischemia–reperfusion

Acknowledgements

We would like to thank the Department of Clinical Research (grant no. DCR/P2/2007), Singapore General Hospital and the Office of Life Science (grant no. R-184-000-074-712), National University of Singapore, Singapore.

References (37)

  • BaskarR. et al.

    Effects of low and high LET radiations on bystander human lung fibroblast cell survival

    Int. J. Radiat. Biol.

    (2007)
  • BeauchampR.O. et al.

    A critical review of the hydrogen sulfide toxicity

    Crit. Rev. Toxicol.

    (1984)
  • BenavidesG.A. et al.

    Hydrogen sulfide mediates the vasoactivity of garlic

    Proc. Natl. Acad. Sci. U. S. A.

    (2007)
  • BrooksG. et al.

    Non-steroidal ant-inflammatory drugs (NSAIDs) inhibit vascular smooth muscle cell proliferation via differential effects on the cell cycle

    J. Pharmacy and Pharmacol.

    (2003)
  • BunzF. et al.

    Requirement for p53 and p21 to sustain G2 arrest after DNA damage

    Science

    (1998)
  • DzauV.J. et al.

    Vascular proliferation and atherosclerosis: new perspectives and therapeutic strategies

    Nat. Med.

    (2002)
  • ElrodJ.W. et al.

    Hydrogen sulfide attenuates myocardial ischemia–reperfusion injury by preservation of mitochondrial function

    Proc. Natl. Acad. Sci. U. S. A.

    (2007)
  • GizardF. et al.

    PPARα inhibits vascular smooth muscle cell proliferation underlying initial hyperplasia by inducing the tumor suppressor p16INK4a

    J. Clin. Invest.

    (2005)
  • Cited by (80)

    • Hydrogen sulfide signaling in regulation of cell behaviors

      2020, Nitric Oxide - Biology and Chemistry
    View all citing articles on Scopus
    View full text