Modifications in low-density lipoprotein receptor expression affects Cyclosporin A cellular uptake and cytotoxicity.
The purpose of this study was to test the effect of modulating the expression of the human low-density lipoprotein receptor (LDLr) in human embryonic kidney (293T) cells on Cyclosporin A (CsA) cellular uptake and CsA-mediated cytotoxicity. LDLr expression was modulated using RNA interference (RNAi) and an LDLr overexpression plasmid. One of the small-interfering RNA (siRNA) constructs, LDLr-792, showed a 60% decrease in LDLr protein expression. The downregulation effect was specific as transfection with an annexin V (AxV) siRNA construct did not decrease LDLr expression levels. AxV and ABCA1 expression levels were not affected in the cells transfected with LDLr-792 (LDLr(LOW) cells) compared to the controls. At a functional level, fluorescent low-density lipoprotein (LDL) (DiI-LDL) internalization in the LDLr(LOW) cells was decreased (30%) compared to control cells. We tested the dose-dependent cytotoxicity induced by CsA using a respiration assay. We found a decrease in CsA-mediated cytotoxicity in the range of CsA doses studied (1-10 microg/mL) in the LDLr(LOW) cells compared to the pSHAG-transfected cells, reaching a statistical significance at 10 microg/mL CsA. At higher CsA doses we found a significant decrease in LDLr expression. When the control and LDLr(LOW) cells were treated with another cytotoxic drug, gentamycin, there was no difference in the cell viability, suggesting that this effect is specific for CsA. We confirmed the association of LDLr expression levels with CsA uptake by overexpressing the LDLr. The LDLr overexpressing cells showed an enhanced uptake of radiolabelled CsA. Taken together these results suggest that CsA internalization and cytotoxicity are affected by the LDL receptor expression levels.
Leon
Carlos
C
Division of Pharmaceutics and Biopharmaceutics, Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver British Columbia, Canada V6T 1Z3.
Jia
Jessica
J
Qiu
Guosong
G
Hill
John S
JS
Wasan
Kishor M
KM
D002460:Cell Line; D019069:Cell Respiration / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D016572:Cyclosporine / Q000378:metabolism* / Q000633:toxicity*; D004305:Dose-Response Relationship, Drug; D005839:Gentamicins / Q000378:metabolism / Q000633:toxicity; D006801:Humans; D007166:Immunosuppressive Agents / Q000378:metabolism* / Q000633:toxicity*; D008077:Lipoproteins, LDL / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D011973:Receptors, LDL / Q000235:genetics / Q000378:metabolism*; D014162:Transfection