Radioprotective effect on HepG2 cells of low concentrations of cobalt chloride: induction of hypoxia-inducible factor-1 alpha and clearance of reactive oxygen species

Abstract

It has been found that low doses of certain toxicants might generate a protective response to cellular damage. Previous data have shown that elevated doses of cobalt (Co) induce injury to cells and organisms or result in radiological combined toxicity. Whether low doses of Co generate a protective effect or not, however, remains controversial. In this study, we investigated the effect and mechanism of action of low dose cobalt chloride (CoCl2, 100 μM) on the viability of irradiated cells. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay was used to observe the radio-sensitivity of HepG2 cells under different pretreatments. The alteration of intracellular DNA damage was further measured using micronucleus (MN) assay. Levels of hypoxia inducible factor-1α (HIF-1α) expression and its target gene, EPO, were monitored by western blot and reverse transcription polymerase chain reaction (RT-PCR), respectively, and intracellular reactive oxygen species (ROS) content was determined by 2′,7′-dichlorofluorescein diacetate (DCFH-DA) probe staining. Our results show that low dose CoCl2does not influence HepG2 cell viability, but induces the expression of HIF-1α, followed by increased radio-resistance. Additionally, cells treated with HIF-1α siRNA retained a partial refractory response to irradiation concomitant with a marked reduction in intracellular ROS. The change of MN further indicated that the reduction of DNA damage was confirmed with the alteration of ROS. Our results demonstrate that low dose CoCl2may protect cells against irradiative harm by two mechanisms, namely HIF-1α expression and ROS clearance.

Paclitaxel pretreatment overcomes hypoxia inducible factor-1α-induced radioresistance acquisition of human hepatoma and lung adenocarcinoma cells

“…We uniquely identified a subclone variant, J5 cells, that overexpressed HIF-1α protein normoxically, and the relative tolerance of these cells against a single fraction of radiation (20 Gy) was also overwhelmingly strong compared with the non-HIF-1α-expressing counterparts (Figure 1). This finding is consistent with a recent report by Jin et al [21], in which a forced expression of HIF-1α by CoCl 2 conferred radioprotection of hepatoma Hep G2 cells. We also reported previously that neutralization of normoxically overexpressed HIF-1α by YC-1, which is a naturally occurring HIF-1α inhibitor, sensitized J5 cells for efficacious eradication against a reactive oxygen species (ROS)-producing anti-cancer compound, arsenic trioxide (ATO) [22], which is similar to the ROS production manifested by RT.…”

Oleanolic acid enhances the radiosensitivity of tumor cells under mimetic hypoxia through the reduction in intracellular GSH content and HIF-1α expression

“…It was demonstrated that OA might decrease the level of GSH via the inhibition of γ-GCS activity in hypoxic tumor cells. According to our previous study and other data, there was a higher level of cellular GSH in hypoxic cells or mimetic hypoxic cells compared with that in aerobic cells, resulting in the refractoriness of cells to irradiation (36,39,40). Therefore, the combination of OA and radiation to effectively destroy hypoxic tumor cells is strongly correlated with the inhibition of intracellular GSH biosynthesis.…”

“…Therefore, CoCl 2 can be universally used as a chemical reagent that induces biochemical and molecular responses similar to those observed under a hypoxic condition (37,38). Our previous results also showed that similar to hypoxia, CoCl 2 enhanced cellular radioresistance and increased the levels of HIF-1α (36). We selected two different concentrations of OA, with no obvious influence on cell viability, to carry out the present experiment.…”

“…Based on a large number of experimental data, preliminary exposure to mimetic hypoxia with CoCl 2 inducing a similar to real hypoxic condition may increase the tolerance against subsequent injury of other biological and physicochemical factors including chemotherapeutic drugs, ionizing radiation and tert-butyl-hydroperoxide-induced oxidative stress (34)(35)(36). Therefore, CoCl 2 can be universally used as a chemical reagent that induces biochemical and molecular responses similar to those observed under a hypoxic condition (37,38).…”

The role of HIF-1α in chemo-/radioresistant tumors

“…Shenoy et al 41 showed that HIF-1α enhanced DNA repair through upregulating XPA, which leads to cisplatin resistance in testicular germ cell tumors ( Table 1 ). In a study about the mechanism of chemo-/radioresistance in hepatocellular carcinoma, Jin et al 42 ( Table 1 ) demonstrated that HIF-1α inhibited the formation of both radiotherapy-induced DSBs and SSBs. Klein et al 43 ( Table 1 ) suggested that the HIF-1α-activated DNA damage repair pathway also has an emerging role in chemo-/radioresistance in gastric cancer.…”