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Effects of dexamethasone on human trabecular meshwork cells in vitro
Ashish Sharma1, Alammaprabhu Jayaprakash Patil, Saffar Mansoor
1Department of Ophthalmology, Gavin Herbert Eye Institute, University of California, Irvine, CA, USA. drashish79@hotmail.com
Purpose:
To study the effects of dexamethasone sodium phosphate (Dex) on human trabecular meshwork (HTM) cells in vitro.
Methods:
HTM cells were treated with Dex 2 mg/ml, 1 mg/ml, 0.5 mg/ml, 0.25 mg/ml, 0.1 mg/ml, or 0.05 mg/ml for 24 h. Cell viability was measured by a trypan blue exclusion test. Caspase-3/7, -8, -9 and -12 activities were measured by fluorochrome assays as mean signal intensity (msi) to assess apoptosis. Mitochondrial dehydrogenase activity was determined by a WST assay to quantify mitochondrial damage.
Results:
Mean cell viabilities of HTM cells exposed to Dex at the higher doses of 2 mg/ml, 1 mg/ml, and 0.5 mg/ml were reduced: 11.9 % ± 3.5 (P < 0.001), 31.2 % ± 3.2 (P < 0.001), and 76.6 % ± 4.4 (P < 0.01). At the lower doses of 0.25 mg/ml, 0.1 mg/ml or 0.05 mg/ml, no significant cell viability reductions were seen: 96.3 % ± 0.7 (P > 0.05), 95.3 % ± 2.5 (P > 0.05) and 93.8 % ± 2.3 (P > 0.05), respectively compared to untreated HTM cells (97.0 % ± 1.9). Caspase-3/7 activity (msi) of HTM cells exposed to Dex 2, 1 or 0.5 mg/ml was 21068 ± 2498 (P < 0.001), 26994 ± 3104 (P < 0.001) and 20416 ± 1150 (P < 0.001) compared to untreated HTM cells 1148 ± 803. Caspase-9 activity (msi) of HTM cells after exposure to Dex 2, 1 or 0.5 mg/ml was 14188 ± 1203 (P < 0.001), 13256 ± 1564 (P < 0.001) and 15041 ± 1584 (P < 0.001) compared to untreated HTM cells 1748 ± 524. The lower doses of Dex did not significantly increase caspase-3/7 or -9 activities. There were no increases for caspase-8 or -12 activities at any of the tested Dex doses. The WST assay showed mitochondrial dehydrogenase activities of 14.3 ± 0.7 (P < 0.001), 9.6 ± 0.3 (P < 0.001) and 56.0 ± 7.6 (P < 0.001) at 2 mg/ml, 1 mg/ml and 0.5 mg/ml Dex compared to untreated HTM cells (186.1 ± 15.0).
Conclusions:
Dex at 0.25, 0.1 and 0.05 mg/ml clinical dose did not cause significant reduction in cell viability, increased apoptosis, or mitochondrial dysfunction of HTM cells in vitro. At high doses (2, 1 or 0.5 mg/ml) Dex caused apoptosis via mitochondrial pathways.
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