Differential cytotoxic effects of 7-dehydrocholesterol-derived oxysterols on cultured retina-derived cells: Dependence on sterol structure, cell type, and density

Abstract

Tissue accumulation of 7-dehydrocholesterol (7DHC) is a hallmark of Smith-Lemli-Opitz Syndrome (SLOS), a human inborn error of the cholesterol (CHOL) synthesis pathway. Retinal 7DHC-derived oxysterol formation occurs in the AY9944-induced rat model of SLOS, which exhibits a retinal degeneration characterized by selective loss of photoreceptors and associated functional deficits, Müller cell hypertrophy, and engorgement of the retinal pigment epithelium (RPE) with phagocytic inclusions. We evaluated the relative effects of four 7DHC-derived oxysterols on three retina-derived cell types in culture, with respect to changes in cellular morphology and viability. 661W (photoreceptor-derived) cells, rMC-1 (Müller glia-derived) cells, and normal diploid monkey RPE (mRPE) cells were incubated for 24 h with dose ranges of either 7-ketocholesterol (7kCHOL), 5,9-endoperoxy-cholest-7-en-3β,6α-diol (EPCD), 3β,5α-dihydroxycholest-7-en-6-one (DHCEO), or 4β-hydroxy-7-dehydrocholesterol (4HDHC); CHOL served as a negative control (same dose range), along with appropriate vehicle controls, while staurosporine (Stsp) was used as a positive cytotoxic control. For 661W cells, the rank order of oxysterol potency was: EPCD > 7kCHOL >> DHCEO > 4HDHC ≈ CHOL. EC50 values were higher for confluent vs. subconfluent cultures. 661W cells exhibited much higher sensitivity to EPCD and 7kCHOL than either rMC-1 or mRPE cells, with the latter being the most robust when challenged, either at confluence or in sub-confluent cultures. When tested on rMC-1 and mRPE cells, EPCD was again an order of magnitude more potent than 7kCHOL in compromising cellular viability. Hence, 7DHC-derived oxysterols elicit differential cytotoxicity that is dose-, cell type-, and cell density-dependent. These results are consistent with the observed progressive, photoreceptor-specific retinal degeneration in the rat SLOS model, and support the hypothesis that 7DHC-derived oxysterols are causally linked to that retinal degeneration as well as to SLOS.

Streamlined duplex live-dead microplate assay for cultured cells

“…The methods outlined here are amenable, however, to the inclusion of inhibitors, antioxidants, and other protective reagents before, during, or after exposure to treatments that generate mechanisms leading to various modes of cell death. Additionally, a time course experiment could conceivably isolate different steps in the cell death process; this strategy is quite straightforward to design using the CaAM and SO reagents, such as was carried out previously for the cytotoxic oxysterol 7kCHOL (Pfeffer et al, 2016). In this latter example, although the major phase of measurable loss of cell viability occurred by 6 h of treatment with a single concentration of 7kCHOL, routine investigations used a 24-h endpoint.…”

“…The rMC-1 cells were generously provided by Dr. Vijay Sarthy (Northwestern University School of Medicine, Evanston, IL, USA). During the course of investigations described herein, both cell lines were extensively characterized genomically, and also by analysis of expressed signature genes and proteins (Pfeffer et al, 2016). Cell lines were utilized between passages 20 and 50.…”

“…The formulation of the growth medium for retinal neural and glial cell lines routinely used in our laboratory (Pfeffer et al, 2016), including sources of individual components, is given in Table 3. This reduced serum medium resembles Neurobasal medium plus B-27 supplements, developed for neural cell culture in general, (Brewer et al, 1993), using a recent modification (Chen et al, 2008; this reference provides additional details for formulating stocks for most of the medium components in Table 3).…”

“…We previously showed that in experiments using subconfluent 661W cells, 40 – 50 nM Stsp was sufficient for a maximal loss of viability, as measured by several assays, including CaAM and SO (Pfeffer et al, 2016). After passive internalization of hydrophobic CaAM through the plasma membrane, viable cells promote the esterase-mediated hydrolysis of CaAM to its fluorescent, more polar and membrane-impermeable product, calcein (Bozyczko-Coyne et al, 1993).…”

The Role of Oxysterols in Human Cancer

“…The pro-apoptotic or cytostatic activity of oxysterols observed in various model cancer cell lines, e.g., murine lymphoma [73], human lymphoma and leukemic cells [74–76], as well as in non-tumor cells, e.g., thymocytes [73], retina-derived cells [77], adipose tissue-derived mesenchymal stem cells [78] and rat and human vascular smooth muscle cells [15, 16], suggests that these effects have universal importance.…”