Oncotarget. 2011 Oct;2(10):761-82.

Lithocholic bile acid selectively kills neuroblastoma cells, while sparing normal neuronal cells.

Goldberg AA, Beach A, Davies GF, Harkness TA, Leblanc A, Titorenko VI.

Source

Department of Biology, Concordia University, Montreal, Quebec H4B 1R6, Canada.

Abstract

Aging is one of the major risk factors of cancer. The onset of cancer can be postponed by pharmacological and dietary anti-aging interventions. We recently found in yeast cellular models of aging that lithocholic acid (LCA) extends longevity. Here we show that, at concentrations that are not cytotoxic to primary cultures of human neurons, LCA kills the neuroblastoma (NB) cell lines BE(2)-m17, SK-n-SH, SK-n-MCIXC and Lan-1. In BE(2)-m17, SK-n-SH and SK-n-MCIXC cells, the LCA anti-tumor effect is due to apoptotic cell death. In contrast, the LCA-triggered death of Lan-1 cells is not caused by apoptosis. While low concentrations of LCA sensitize BE(2)-m17 and SK-n-MCIXC cells to hydrogen peroxide-induced apoptotic cell death controlled by mitochondria, these LCA concentrations make primary cultures of human neurons resistant to such a form of cell death. LCA kills BE(2)-m17 and SK-n-MCIXC cell lines by triggering not only the intrinsic (mitochondrial) apoptotic cell death pathway driven by mitochondrial outer membrane permeabilization and initiator caspase-9 activation, but also the extrinsic (death receptor) pathway of apoptosis involving activation of the initiator caspase-8. Based on these data, we propose a mechanism underlying a potent and selective anti-tumor effect of LCA in cultured human NB cells. Moreover, our finding that LCA kills cultured human breast cancer and rat glioma cells implies that it has a broad anti-tumor effect on cancer cells derived from different tissues and organisms.

PMID:: 21992775; [PubMed - in process]
PMCID:: PMC3248158

Free PMC Article

Images from this publication.See all images (8) Free text

Figure 3

LCA significantly increases the activity of the initiator caspase-9 in cultured human NB cell lines BE(2)-m17 and SK-n-MCIXC

Specific activity of caspase-9 was measured as described in Materials and Methods and expressed in picomoles of fluorescent compound 7-amino-4-trifluoromethyl coumarin released per microgram of protein per minute, based on the linear range of the curve. Data are presented as means ± SD (n = 3-4); ***p<0.001.

Lithocholic bile acid selectively kills neuroblastoma cells, while sparing normal neuronal cells

Oncotarget. Oncotarget;2(10):761-782.

Figure 7

LCA increases activity of the initiator caspase-8 in cultured human NB cell lines BE(2)-m17 and SK-n-MCIXC (but not in Lan-1)

Specific activity of caspase-8 was measured as described in Materials and Methods and expressed in picomoles of fluorescent compound 7-amino-4-trifluoromethyl coumarin released per microgram of protein per minute, based on the linear range of the curve. Data are presented as means ± SD (n = 3-4); ***p<0.001.

Lithocholic bile acid selectively kills neuroblastoma cells, while sparing normal neuronal cells

Oncotarget. Oncotarget;2(10):761-782.

Figure 4

In cultured human NB cell lines BE(2)-m17 and SK-n-MCIXC (but not in Lan-1), LCA causes mitochondrial fragmentation and triggers the dissipation of the electrochemical potential across the inner mitochondrial membrane

A. Mitochondrial morphology in NB cells treated with LCA or remained untreated was visualized using MitoTracker Red CMXRos as described in Materials and Methods. Cells were viewed using fluorescence microscopy. B. The percentage of cells displaying fragmented mitochondria was calculated. C. The mitochondrial membrane potential (∆Ψ) in NB cells treated with LCA or remained untreated was measured using tetramethylrhodamine ethyl ester (TMRE) as described in Materials and Methods; the extent of reversible sequestration of TMRE by mitochondria is proportional to the value of ∆Ψ. Cells were incubated with 50 nM TMRE for 20 min and directly viewed using fluorescence microscopy. D. The percentage of TMRE-positive cells displaying a detectable level of ∆Ψ was calculated. Data in B and D are presented as means ± SD (n = 3-4).

Lithocholic bile acid selectively kills neuroblastoma cells, while sparing normal neuronal cells

Oncotarget. Oncotarget;2(10):761-782.

Figure 8

A model for a mechanism underlying an anti-tumor effect of LCA in cultured human NB cell lines BE(2)-m17 and SK-n-MCIXC

See text for details. Abbreviations: AC, adenylyl cyclase; BID, BH3-interacting domain death agonist; CD95/Fas (factor-activating Exo S), death receptor; CREB, cAMP-response element binding protein; Csp-1, -3, -6, -8 and -9, caspases-1, -3, -6, -8 and -9; DISC, death-inducing signaling complex; G, a G-protein; JNK, c-Jun-N terminal kinase; LCA, lithocholic acid; MEKK1/2/3, mitogen-activated protein kinase/ERK kinase kinase-1, -2 and -3; MKK4/7, mitogen-activated protein kinase kinase-4 and -7; MOMP, mitochondrial outer membrane permeabilization; PKA, protein kinase A; ROS, reactive oxygen species; TGR5, a member of the G-protein coupled receptor family.

Lithocholic bile acid selectively kills neuroblastoma cells, while sparing normal neuronal cells

Oncotarget. Oncotarget;2(10):761-782.

Figure 6

LCA increases activity of the executioner caspase-6 in cultured human NB cell lines BE(2)-m17 and SK-n-MCIXC (but not in Lan-1) by stimulating a proteolytic conversion of a zymogen pro-caspase-6 form into its large and small subunits known to assemble into an enzymatically active heterotetramer consisted of the two of each subunits

A. Specific activity of caspase-6 was measured as described in Materials and Methods and expressed in picomoles of fluorescent compound 7-amino-4-trifluoromethyl coumarin released per microgram of protein per minute. B. Pro-caspase-6 cleavage assay was carried out as described in Materials and Methods. The anti-human-caspase-6 mouse monoclonal IgG1 clone B93-4 antibodies raised against amino acids 271-285 (Pharmingen) were used; these antibodies specifically recognize the large (p20) and small (p10) subunits of an enzymatically active form of caspase-6. Abbreviations: FL, a full-length zymogen form of pro-caspase-6; p20p10, caspase-6 lacking the pro-domain; p20 and p10, large and small subunits (respectively) of caspase-6 lacking the pro- and linker-domains; ★, an unidentified protein band, perhaps p20-linker (p23), pro-p20 (p23) or pro-p20-linker subunits of caspase-6 [152]. Data in A are presented as means ± SD (n = 3-4); *p<0.05; **p<0.01; ***p<0.001.

Lithocholic bile acid selectively kills neuroblastoma cells, while sparing normal neuronal cells

Oncotarget. Oncotarget;2(10):761-782.

Figure 1

LCA selectively kills all four tested lines of cultured human NB cells - by causing an apoptosis-based death of BE(2)-m17, SK-n-SH and SK-n-MCIXC lines, but triggering a non-apoptotic death of Lan-1 line

In A, the percentage of viable cells was calculated as a portion of their population displaying a detectable level of redox potential which was monitored using the MTT (3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide)-based CellTiter 96 Non-Radioactive Cell Proliferation Assay. In B, the fluorescent dye Hoechst was used to visualize chromatin in various cell cultures and the percentage of viable non-apoptotic cells carrying intact, non-fragmented nuclei containing non-condensed chromatin was calculated; dead apoptotic cells carried fragmented nuclei containing condensed chromatin, a hallmark event of apoptotic death. A. LCA kills NB cell lines BE(2)-m17, SK-n-SH, SK-n-MCIXC and Lan-1 if used at concentrations that are not cytotoxic or only mildly cytotoxic (as in case of Lan-1) to primary cultures of human neurons. B. The NB cell line SK-n-MCIXC is the most sensitive to LCA-induced apoptotic death; two other lines, BE(2)-m17 and SK-n-SH, exhibit much higher sensitivity to LCA-induced apoptotic death than primary cultures of human neurons or the NB cell line Lan-1. Data are presented as means ± SD (n = 4-6).

Lithocholic bile acid selectively kills neuroblastoma cells, while sparing normal neuronal cells

Oncotarget. Oncotarget;2(10):761-782.

Figure 5

LCA increases activity of the executioner caspase-3 in cultured human NB cell lines BE(2)-m17 and SK-n-MCIXC (but not in Lan-1) by stimulating a proteolytic conversion of its zymogen pro-caspase-3 form into its enzymatically active 17 kDa form

The anti-tumor effect of LCA in BE(2)-m17 and SK-n-MCIXC cell lines is due in part to its ability to increase caspase-3 activity. A and D. Specific activity of caspase-3 was measured in NB cells treated with LCA and/or z-DEVD-fmk (a potent and specific inhibitor of caspase-3) or remained untreated as described in Materials and Methods and expressed in picomoles of fluorescent compound 7-amino-4-trifluoromethyl coumarin released per microgram of protein per minute. B and C. Pro-caspase-3 cleavage assay was carried out as described in Materials and Methods. E. The fluorescent dye Hoechst was used to visualize chromatin in NB cell cultures treated with LCA and/or z-DEVD-fmk or remained untreated. For each cell culture, the percentage of viable non-apoptotic cells carrying intact, non-fragmented nuclei containing non-condensed chromatin was calculated; dead apoptotic cells carried fragmented nuclei containing condensed chromatin, a hallmark event of apoptotic death. Data in A, D and E are presented as means ± SD (n = 3-4); **p<0.01; ***p<0.001.

Lithocholic bile acid selectively kills neuroblastoma cells, while sparing normal neuronal cells

Oncotarget. Oncotarget;2(10):761-782.

Figure 2

LCA increases sensitivity of cultured human NB cells to hydrogen peroxide-induced apoptotic cell death

A. If used at concentrations below 100 μM, LCA protects primary cultures of human neurons against mitochondria-controlled apoptosis induced in response to exogenously added 0.1 mM hydrogen peroxide. B and C. In contrast, if used at concentrations below 100 μM, LCA greatly enhances the susceptibility of cultured human NB cell lines BE(2)-m17 (B) and SK-n-MCIXC (C) to such hydrogen peroxide-induced form of cell death. While 75 μM LCA causes apoptotic death of all or most of the cells of BE(2)-m17 and SK-n-MCIXC exposed to 0.1 mM hydrogen peroxide (B and C, respectively), in this concentration it increases the resistance of primary cultures of human neurons to such form of cell death (A). Nuclear fragmentation and chromatin condensation were visualized with the fluorescent dye Hoechst and were used as a measure of the efficacy of hydrogen peroxide-induced apoptotic cell death. Each panel shows two datasets for the same cell type. One dataset is for cell aliquots treated with 0.1 mM hydrogen peroxide, whereas the other dataset is for cell aliquots remained untreated. For each cell culture, the percentage of viable non-apoptotic cells carrying intact, non-fragmented nuclei containing non-condensed chromatin was calculated. Data are presented as means ± SD (n = 3-5).

Lithocholic bile acid selectively kills neuroblastoma cells, while sparing normal neuronal cells

Oncotarget. Oncotarget;2(10):761-782.

LinkOut - more resources

Full Text Sources

Impact Journals, LLC
PubMed Central
PubMed Central Canada
UK PubMed Central

PubMed

Display Settings:

Send to:

Lithocholic bile acid selectively kills neuroblastoma cells, while sparing normal neuronal cells.

Source

Abstract

LinkOut - more resources

Full Text Sources

Supplemental Content

Related citations

Related information

Recent activity

Simple NCBI Directory

Getting Started

Resources

Popular

Featured

NCBI Information