Effect of curcumin on proliferation of human retinal endothelial cells under in vitro conditions.
OBJECTIVE
To investigate the effect of high glucose on the proliferation of human retinal endothelial cells (HRECs) and to elucidate the possible mechanisms of antiangiogenic activity of curcumin, a diferuloylmethane.


METHODS
Human retinal endothelial cells were isolated from the retinal tissue obtained from human donors and the culture system was established. The effect of curcumin on the proliferation of primary HRECs in the presence of low and high glucose was measured by MTT and thymidine uptake assays. Apoptosis was assessed by TUNEL assay and other adjuvant tools. Effect of curcumin on phorbol ester stimulated intracellular reactive oxygen species (ROS) generation in high glucose conditions was assessed by fluorescence assay. Finally, semiquantitative RT-PCR and Western blot analysis was performed to measure VEGF mRNA production and VEGF induced PKC-betaII translocation, respectively in the presence and absence of curcumin.


RESULTS
HREC culture was established successfully at passages 3 and 4 at 80% confluence. Curcumin effectively inhibited endothelial cell proliferation in a dose-dependent manner. At a concentration of 10 microM, curcumin significantly inhibited HREC proliferation in high-glucose-treated cells, as verified by both MTT and thymidine uptake assay. Curcumin also showed a significant (P = 0.03) reduction of intracellular ROS generation in HRECs. RNA expression studies showed that curcumin had an inhibitory effect on the glucose-induced VEGF mRNA expression. In addition, VEGF-mediated, membrane-associated changes in the PKC-betaII translocation in HRECs was inhibited by 31% on treatment with 10 microM curcumin.


CONCLUSIONS
These data suggest an underlying mechanism whereby curcumin induces the apoptosis in HRECs by the regulation of intracellular ROS generation, VEGF expression and release, and VEGF-mediated PKC-betaII translocation.
Premanand
Chinnaraj
C
Madras Diabetes Research Foundation, Gopalapuram, Chennai, India.
Rema
Mohan
M
Sameer
Mahmood Z
MZ
Sujatha
Mohan
M
Balasubramanyam
Muthusamy
M
D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D018929:Cell Culture Techniques; D049109:Cell Proliferation / Q000187:drug effects*; D002470:Cell Survival; D003474:Curcumin / Q000494:pharmacology*; D004247:DNA / Q000096:biosynthesis; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D004797:Enzyme-Linked Immunosorbent Assay; D005947:Glucose / Q000494:pharmacology; D006801:Humans; D011493:Protein Kinase C / Q000378:metabolism; D064546:Protein Kinase C beta; D012333:RNA, Messenger / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D012171:Retinal Vessels / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism
D000970:Antineoplastic Agents; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D004247:DNA; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D003474:Curcumin; D005947:Glucose