Ectopic expression of clusterin/apolipoprotein J or Bcl-2 decreases the sensitivity of HaCaT cells to toxic effects of ropivacaine.
Local anesthetics inhibit cell proliferation and induce apoptosis in various cell types. Ropivacaine, a unique, novel tertiary amine-type anesthetic, was shown to inhibit the proliferation of several cell types including keratinocytes. We found that Ropivacaine could inhibit the proliferation and induce apoptosis in an immortalized human keratinocyte line, HaCaT, in a dose- and time-dependent manner and with the deprivation of serum. The dose-dependent induction of apoptosis by ropivacaine was demonstrated by DNA fragmentation analysis and the proteolytic cleavage of a caspase-3 substrate-poly (ADP-ribose) polymerase (PARP). In addition, ropivacaine downregulated the expression of clusterin/ apoliporotein J, a protein with anti-apoptotic properties, in a dose-dependent manner, which well correlated with the induction of apoptosis of HaCaT cells. To investigate the role of clusterin/apoliporotein J in ropivacaine-induced apoptosis, HaCaT cells overexpressing clusterin/apoliporotein J were generated and compared to cells expressing the well established anti-apoptotic Bcl-2 protein. Ectopic overexpression of the secreted form of clusterin/apoliporotein J or Bcl-2 decreased the sensitivity of HaCaT cells to toxic effects of ropivacaine as demonstrated by DNA fragmentation, the proteolytic cleavage of PARP and by a reduction in procaspase-3 expression. Furthermore, the downregulation of endogenous clusterin/apolipoprotein J levels by ropivacaine suggested that this might be one mechanism by which ropivacaine induced cell death in HaCaT cells. In conclusion, the ability of ropivacaine to induce antiproliferative responses and to suppress the expression of the anti-apoptotic protein clusterin/apolipoprotein J, combined with previously reported anti-inflammatory activity and analgesic property of the drug, suggests that ropivacaine may have potential utility in the local treatment of tumors.
Kontargiris
Evangelos
E
Laboratory of Experimental Physiology, University of Ioannina Medical School, 45110 Ioannina, Greece.
Kolettas
Evangelos
E
Vadalouca
Athina
A
Trougakos
Ioannis P
IP
Gonos
Efstathios S
ES
Kalfakakou
Vasiliki
V
D000577:Amides / Q000633:toxicity*; D017209:Apoptosis / Q000187:drug effects; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D051152:Clusterin; D004307:Dose-Response Relationship, Radiation; D006023:Glycoproteins / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D015603:Keratinocytes / Q000187:drug effects / Q000378:metabolism*; D018832:Molecular Chaperones / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D000077212:Ropivacaine
D000577:Amides; C495242:CLU protein, human; D051152:Clusterin; D006023:Glycoproteins; D018832:Molecular Chaperones; D019253:Proto-Oncogene Proteins c-bcl-2; D000077212:Ropivacaine