Ectopic expression of clusterin/apolipoprotein J or Bcl-2 decreases the sensitivity of HaCaT cells to toxic effects of ropivacaine

Abstract

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.

The effect of two drug delivery systems in ropivacaine cytotoxicity and cytokine release by human keratinocytes and fibroblasts

“…Apoptosis was evident only with 1000 lM ropivacaine, being the cell viability reduced by 50% with 2000 lM ropivacaine. [33] Similar results were observed in the present study, as HaCaT viability was reduced by all ropi formulations at 6 h of exposition, remaining low at the 24-hour period. In addition, the used ropi concentrations did not change the apoptosis rate.…”

“…Exposure to 250 μ m ropivacaine for 24 h reduced the HaCaT proliferation rate. Apoptosis was evident only with 1000 μ m ropivacaine, being the cell viability reduced by 50% with 2000 μ m ropivacaine . Similar results were observed in the present study, as HaCaT viability was reduced by all ropi formulations at 6 h of exposition, remaining low at the 24‐hour period.…”

Hypoxia: a critical regulator of early human tendinopathy

“…Our study now reveals HIF-1α expression in early tendinopathy biopsies. Clusterin46 and Bcl-247 have been functionally implicated in DNA repair, cell cycle regulation, while both play a prosurvival role during cell death 48. Their abundant expression in early tendinopathy biopsies and overexpression under hypoxic conditions at both the protein and mRNA level strongly supports the concept of hypoxia as a regulator of apoptosis.…”

Se-Methylselenocysteine Inhibits Apoptosis Induced by Clusterin Knockdown in Neuroblastoma N2a and SH-SY5Y Cell Lines

“…Clu can inhibit NF-κB signaling through stabilization of IκBs (inhibitor of κB) [ 23 ]. Over-expression of human Clu has been reported to protect human epidermal keratinocyte (HaCaT) cells from ropivacaine-induced apoptosis [ 24 ]. MSC is a new type of organic selenium compound, which is prevalent in broccoli [ 25 ] and garlic [ 26 ].…”