Unconjugated bilirubin induces apoptosis in colon cancer cells by triggering mitochondrial depolarization

Abstract

Bilirubin is the principal end product of heme degradation. Prompted by epidemiologic analyses demonstrating an inverse correlation between serum bilirubin levels and cancer mortality, we examined the effect(s) of bilirubin on the growth and survival of colon adenocarcinoma cells. Adenocarcinoma cell monolayers were treated with bilirubin over a range of bilirubin:BSA molar ratios (0 -0.6), and viability was assessed colorimetrically. Apoptosis was characterized by TUNEL assay, annexin V staining and caspase-3 activation. The mechanism(s) by which bilirubin induces apoptosis was investigated by Western blotting for cytochrome c release, assaying for caspase-8 and caspase-9 activation and for mitochondrial depolarization by JC-1 staining. The direct effect of bilirubin on the membrane potential of isolated mitochondria was evaluated using light-scattering and fluorescence techniques. Bilirubin decreased the viability of all colon cancer cell lines tested in a dose-dependent manner. Cells exhibited substantial apoptosis when exposed to bilirubin concentrations ranging 0 -50 M, as demonstrated by an 8-to 10-fold increase in TUNEL and annexin V staining and in caspase-3 activity. Bilirubin treatment evokes specific activation of caspase-9, enhances cytochrome c release into the cytoplasm and triggers the mitochondrial permeability transition in colon cancer monolayers. Additionally, bilirubin directly induces the depolarization of isolated rat liver mitochondria, an effect that is not inhibited by cyclosporin A. Bilirubin stimulates apoptosis of colon adenocarcinoma cells in vitro through activation of the mitochondrial pathway, apparently by directly dissipating mitochondrial membrane potential. As this effect is triggered at concentrations normally present in the intestinal lumen, we postulate a physiologic role for bilirubin in modulating colon tumorigenesis.

Unconjugated Bilirubin Is a Novel Prognostic Biomarker for Nasopharyngeal Carcinoma and Inhibits Its Metastasis via Antioxidation Activity

“…Previous studies suggested that UCB may inhibit cell growth and induce apoptosis in cancer cells (28,29). However, our results suggested that 20 mmol/L UCB does not significantly affect the growth and apoptosis of NPC cells both in vitro and in vivo (Supplementary Fig.…”

“…A small number of studies have suggested that UCB may inhibit proliferation and induce apoptosis in cancer cells (28,29). However, we did not observe an effect on NPC cells until the concentration of UCB reached 100 mmol/L (Supplementary Fig.…”

“…We speculate the reason for this result is that the sensitivity to the toxicity of UCB is different among tumors. Even within one tumor type, different cell lines are reported to have disparate sensitivities (29). Further studies need to be performed to determine why different tumors have disparate sensitivities to UCB.…”

Cytochrome P450 2A5 and bilirubin: Mechanisms of gene regulation and cytoprotection

“…Previous studies have shown that caspase-8 activity was significantly induced by BR treatment in Hepa 1c1c7 and rat neurons, suggesting BR-induced apoptosis is mediated by the extrinsic apoptosis pathway (Seubert et al, 2002;Vaz et al, 2011). However, caspase-8 induction appears to be cell-type specific, hence it remains unclear whether BR acts as a ligand for the death receptor or whether caspase-8 activity is increased indirectly by BR via oxidative stress in cellular organelles (Keshavan et al, 2004).…”

“…Fas) may be involved (Seubert et al, 2002;Vaz et al, 2011). However, BR-induced apoptosis via extrinsic pathway remains to be confirmed as the induction may be cell type-specific (Keshavan et al, 2004). BRinduced mitochondrial apoptotic pathway is also the result of production of lipid peroxides, hydrogen peroxide, and hydroxyl radical, as well as a decrease in glutathione content and reduction of the mitochondrial membrane (Kumar et al, 2008;Brites and Brito, 2012).…”