Bioluminescence‐based cytotoxicity assay for simultaneous evaluation of cell viability and membrane damage in human hepatoma HepG2 cells

Abstract

We have developed a bioluminescence-based non-destructive cytotoxicity assay in which cell viability and membrane damage are simultaneously evaluated using Emerald luciferase (ELuc) and endoplasmic reticulum (ER)-targeted copepod luciferase (GLuc-KDEL), respectively, by using multi-integrase mouse artificial chromosome (MI-MAC) vector. We have demonstrated that the time-dependent concentration response curves of ELuc luminescence intensity and WST-1 assay, and GLuc-KDEL luminescence intensity and lactate dehydrogenase (LDH) activity in the culture medium accompanied by cytotoxicity show good agreement in toxicant-treated ELuc- and GLuc-KDEL-expressing HepG2 stable cell lines. We have clarified that the increase of GLuc-KDEL luminescence intensity in the culture medium reflects the type of cell death, including necrosis and late apoptosis, but not early apoptosis. We have also uncovered a strong correlation between GLuc-KDEL luminescence intensity in the culture medium and the extracellular release of high mobility group box 1 (HMGB1), a representative damage-associated molecular pattern (DAMP) molecule. The bioluminescence measurement assay using ELuc and GLuc-KDEL developed in this study can simultaneously monitor cell viability and membrane damage, respectively, and the increase of GLuc-KDEL luminescence intensity in the culture medium accompanied by the increase of cytotoxicity is an index of necrosis and late apoptosis associated with the extracellular release of DAMP molecules.

Bioluminescence Measurement of Time-Dependent Dynamic Changes of CYP-Mediated Cytotoxicity in CYP-Expressing Luminescent HepG2 Cells

“…First, to examine the appropriateness of cytotoxicity assessment by bioluminescence measurement, we conducted non-destructive bioluminescence measurement and the WST-1 assay in parallel using CYP-non-expressing luminescent HepG2 cells (ELuc-HepG2 cells) and CYP-expressing luminescent HepG2 cells (CYPs-ELuc-HepG2 cells). We confirmed that the decrease of bioluminescence intensity well correlated with the increase of cytotoxicity, as reported previously [19][20][21]. Furthermore, we obtained similar concentration-response curves for SDS-(non-selective toxicant) or dimethyl fumarate-(CYP-independent toxicant) treated ELuc-HepG2 and CYPs-ELuc-HepG2 cells, respectively (Figure 2), indicating that the sensitivity of the two cell lines to CYP-independent cytotoxicity is almost the same.…”

“…Next, to verify the sensitivity of the two cell lines to toxicants whose toxicity does not depend on CYP metabolism, we conducted non-destructive bioluminescence measurement and water-soluble tetrazolium-1 (WST-1) assay in parallel ( Figure 2). After ELuc-HepG2 and CYPs-ELuc-HepG2 cells were treated with toxicant, the cells were incubated in the presence of D-luciferin, a bioluminescent substrate for ELuc, for three days and bioluminescence intensity was measured non-destructively thereafter, as reported previously [20,21]. Then, the WST-1 assay was performed using the same cells.…”

“…Hypoxanthine phosphoribosyl transferase-deficient CHO) (JCRB0218, Japanese Collection of Research Bioresources, Osaka, Japan) derived cells were maintained in Ham's F-12 nutrient mixture (FUJIFILM Wako Pure Chemical Corporation) supplemented with 10% fetal bovine serum (FBS; HyClone Laboratories, Logan, UT, USA). CHO cells harboring the CAG-ELuc MAC vector were generated by a specific insertion of the pCAG-ELuc plasmid [19] into the R4 attP site of the MAC vector, as reported previously [21]. HepG2-derived cells (RCB1886, RIKEN BRC, Ibaraki, Japan) were maintained in Dulbecco's modified Eagle's medium (DMEM; FUJIFILM Wako Pure Chemical Corporation) supplemented with 10% FBS (HyClone Laboratories), 4 mM L-glutamine, non-essential amino acids (Gibco-BRL, Grand Island, NY, USA), 1 mM pyruvate, 100 units/mL penicillin G, and 100 µg/mL streptomycin (FUJIFILM Wako Pure Chemical Corporation).…”

“…The bioluminescent reporter gene, luciferase, is broadly employed to quantitatively monitor cellular functions [12][13][14]. Luciferase is widely used for the conventional evaluation of cytotoxicity, where a decrease of bioluminescence intensity accompanied by an increase of cytotoxicity is used as an index [15][16][17][18][19][20][21]. Among available luciferases, beetle luciferases have the advantage of precisely monitoring cytotoxicity, namely, it is possible to measure non-destructively the luminescence of luciferase-expressing cells because Dluciferin (benzothiazole), a bioluminescent substrate for beetle luciferases, is very stable in culture medium and easily penetrates cells [22][23][24].…”

Biophysical, Biochemical, and Cell Based Approaches Used to Decipher the Role of Carbonic Anhydrases in Cancer and to Evaluate the Potency of Targeted Inhibitors

“…Therefore, the level of formazan formation is directly proportional to the amount of released LDH in the medium. This assay is a better indicator of necrotic cell death and cytotoxicity [ 146 , 148 ]. Recent studies in our laboratory show that some sulfonamide inhibitors affect cell growth without inducing cytotoxicity and vice versa (Mboge et al submitted).…”