CoCl₂, a Chemical Inducer of Hypoxia‐Inducible Factor‐1, and Hypoxia Reduce Apoptotic Cell Death in Hepatoma Cell Line HepG2

Abstract

HIF-1 (hypoxia-inducible factor-1) is the major transcription factor that is specifically activated during hypoxia. This transcription factor is composed of two subunits: HIF-1alpha and ARNT (aryl hydrocarbon receptor nuclear translocator). ARNT is constitutively expressed, whereas HIF-1alpha is targeted to proteasome degradation by ubiquitination during normoxia. In hypoxia, HIF-1alpha is stabilized and translocates to the nucleus, where it binds to ARNT. The active HIF-1 induces expression of various genes whose products play an adaptive role to the new conditions induced by hypoxia. Besides the role played by HIF-1 in the adaptation to hypoxia, recent data describe a possible role for HIF-1 in the modulation of apoptosis. According to some authors, hypoxia induces apoptosis. However, it has also been reported that hypoxia could protect cells against apoptotic cell death induced by various agents such as serum deprivation and incubation in the presence of chemotherapy agents. These contradictory data suggest that HIF-1 could display either a proapoptotic or an antiapoptotic role according to the conditions. In order to study how HIF-1 can modulate apoptosis, we studied whether hypoxia or cobalt chloride, a chemical inducer of HIF-1, could influence apoptosis induced by tert-butyl hydroperoxide (t-BHP), serum deprivation, or both in hepatoma cell line HepG2. HepG2 cells were incubated 8 hours under normoxia or hypoxia in the presence of t-BHP with or without CoCl2. CoCl2 reduced the apoptotic death of HepG2 cells induced by t-BHP and serum deprivation, as measured by DNA fragmentation. This effect was confirmed by measurement of the caspase activity. Moreover, hypoxia also prevented t-BHP- or serum deprivation-induced DNA fragmentation and caspase activation-however, to a lower extent than CoCl2. These different data suggest a possible antiapoptotic role of HIF-1. More experiments are needed to define if HIF-1 actually plays an active role in cell death protection and to determine the exact mechanism underlying this effect.

Placental growth factor-1 and epithelial haemato–retinal barrier breakdown: potential implication in the pathogenesis of diabetic retinopathy

“…To study whether the extracellular signal-regulated kinase (ERK, also known as EPHB2) pathway is involved, ARPE-19 cells were starved of serum for 48 h before the experiment and the mitogen-activated protein kinase kinase (MEK) 1/2 inhibitor, UO126 (10 μmol/l; Calbiochem, San Diego, CA, USA) was added 1 h prior to the addition of PLGF-1. To mimic hypoxic conditions, ARPE-19 cell monolayers were incubated with CoCl 2 (10 μmol/l) (Sigma Aldrich, Saint-Quentin Fallavier, France) as previously described [16]. The viability of ARPE-19 cells after exposure to hypoxic conditions for variable periods of time was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.…”

Low-Level Light in Combination with Metabolic Modulators for Effective Therapy of Injured Brain

“…This hypoxic mimetic compound-induced hypoxia and the expression of hypoxia-inducible factor 1α significantly (data not shown). 18 Treatment of the cells with 150 μM CoCl 2 for 48 hours caused almost half of the cells to die ( Figure 1A). However, the deaths were diminished significantly when the cells were exposed to LLL at energy densities ranging from 3 to 10 J/cm 2 after 2 hours of CoCl 2 incubation.…”

“…The cells were plated at a density of~1 × 10 4 cells per well in a 96-well plate, or 5 × 10 4 cells per well in a 24-well plate. The cells were subjected to hypoxia by inclusion of 150 μΜ cobalt chloride (CoCl 2 ) in the medium, a well-known hypoxia-mimetic agent, 18 to which 25 mmol/L glucose, 10 mmol/L pyruvate, or 10 mmol/L lactate was added. The concentration of CoCl 2 led to~50% cell death and was thus selected for the study.…”

ARNT/HIF‐1β links high‐risk 1q21 gain and microenvironmental hypoxia to drug resistance and poor prognosis in multiple myeloma

“…Interestingly, unlike HIF‐1α that accumulates after exposed to bortezomib due to inhibition of its degradation via UPS, HIF‐1β was sharply downregulated after treatment with bortezomib. In this context, cobalt (CoCl 2 ), an approach widely used to mimic hypoxia, stabilizes HIF‐1α through inhibition of its hydroxylation mediated by PHDs and following degradation via UPS 50. However, cobalt failed to induce HIF‐1β expression in MM cells, suggesting that this HIF‐1α inducer might not be an optimal approach to mimic hypoxic microenvironment in MM.…”