Limits...
Regulation of hypoxia inducible factor-1α expression by the alteration of redox status in HepG2 cells.

Jin WS, Kong ZL, Shen ZF, Jin YZ, Zhang WK, Chen GF - J. Exp. Clin. Cancer Res. (2011)

Bottom Line: To further confirm the effect of intracellular redox status on HIF-1α expression, N-acetylcysteine (NAC) was added to culture cells for 8 h before the hypoxia treatment.The results showed that BSO pretreatment down-regulated HIF-1α and the effect was concentration-dependent, on the other hand, the increases of intracellular GSH contents by NAC could partly elevate the levels of HIF-1α expression.The levels of P-Gp (MDR-1) and EPO were concomitant with the trend of HIF-1α expression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Teaching & Research Section of Nuclear Medicine, An-hui Medical University, Hefei, China. wensenjn@139.com

ABSTRACT
Hypoxia inducible factor-1 (HIF-1) has been considered as a critical transcriptional factor in response to hypoxia. It can increase P-glycoprotein (P-Gp) thus generating the resistant effect to chemotherapy. At present, the mechanism regulating HIF-1α is still not fully clear in hypoxic tumor cells. Intracellular redox status is closely correlated with hypoxic micro-environment, so we investigate whether alterations in the cellular redox status lead to the changes of HIF-1α expression. HepG2 cells were exposed to Buthionine sulphoximine (BSO) for 12 h prior to hypoxia treatment. The level of HIF-1α expression was measured by Western blot and immunocytochemistry assays. Reduce glutathione (GSH) concentrations in hypoxic cells were determined using glutathione reductase/5,5'-dithiobis-(2-nitrob-enzoic acid) (DTNB) recycling assay. To further confirm the effect of intracellular redox status on HIF-1α expression, N-acetylcysteine (NAC) was added to culture cells for 8 h before the hypoxia treatment. The levels of multidrug resistance gene-1 (MDR-1) and erythropoietin (EPO) mRNA targeted by HIF-1α in hypoxic cells were further determined with RT-PCR, and then the expression of P-Gp protein was observed by Western blotting. The results showed that BSO pretreatment down-regulated HIF-1α and the effect was concentration-dependent, on the other hand, the increases of intracellular GSH contents by NAC could partly elevate the levels of HIF-1α expression. The levels of P-Gp (MDR-1) and EPO were concomitant with the trend of HIF-1α expression. Therefore, our data indicate that the changes of redox status in hypoxic cells may regulate HIF-1α expression and provide valuable information on tumor chemotherapy.

Show MeSH

Related in: MedlinePlus

The change of HIF-1α proteins in HepG2 cells under hypoxic condition by Western blotting measurement. (A) The representative gel picture was taken from three separate experiments. (B) Compared with hypoxic control, the expression of HIF-1α was reduced in BSO concentration-dependent manner, and the analysis of relative densities showed that there was statistical difference the experimental cells by 100 and 200 μM BSO pretreatment respectively (◆p < 0.05, #p < 0.01). After NAC incubation, the expression of HIF-1α was elevated again, and there were significant difference between the group with 100 μM NAC treatment and that without NAC treatment (▲P < 0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3113749&req=5

Figure 3: The change of HIF-1α proteins in HepG2 cells under hypoxic condition by Western blotting measurement. (A) The representative gel picture was taken from three separate experiments. (B) Compared with hypoxic control, the expression of HIF-1α was reduced in BSO concentration-dependent manner, and the analysis of relative densities showed that there was statistical difference the experimental cells by 100 and 200 μM BSO pretreatment respectively (◆p < 0.05, #p < 0.01). After NAC incubation, the expression of HIF-1α was elevated again, and there were significant difference between the group with 100 μM NAC treatment and that without NAC treatment (▲P < 0.01).

Mentions: HIF-1α protein levels were measured using Western blot after BSO pretreatment. When BSO concentration reached at 50 μM, the down-regulation of HIF-1α expression, under the hypoxia condition, was observed in HepG2 cells. It is then very clear that HIF-1α proteins in hypoxic cells were significantly decreased with BSO concentrations gradually increasing. In addition, the inhibition of HIF-1α expression was reversed by 5 mM NAC supplement. However, we also found that NAC failed to elevate the level of HIF-1α expression inhibited by BSO concentration at 200 μM. These results were shown in Figure 3


Regulation of hypoxia inducible factor-1α expression by the alteration of redox status in HepG2 cells.

Jin WS, Kong ZL, Shen ZF, Jin YZ, Zhang WK, Chen GF - J. Exp. Clin. Cancer Res. (2011)

The change of HIF-1α proteins in HepG2 cells under hypoxic condition by Western blotting measurement. (A) The representative gel picture was taken from three separate experiments. (B) Compared with hypoxic control, the expression of HIF-1α was reduced in BSO concentration-dependent manner, and the analysis of relative densities showed that there was statistical difference the experimental cells by 100 and 200 μM BSO pretreatment respectively (◆p < 0.05, #p < 0.01). After NAC incubation, the expression of HIF-1α was elevated again, and there were significant difference between the group with 100 μM NAC treatment and that without NAC treatment (▲P < 0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3113749&req=5

Figure 3: The change of HIF-1α proteins in HepG2 cells under hypoxic condition by Western blotting measurement. (A) The representative gel picture was taken from three separate experiments. (B) Compared with hypoxic control, the expression of HIF-1α was reduced in BSO concentration-dependent manner, and the analysis of relative densities showed that there was statistical difference the experimental cells by 100 and 200 μM BSO pretreatment respectively (◆p < 0.05, #p < 0.01). After NAC incubation, the expression of HIF-1α was elevated again, and there were significant difference between the group with 100 μM NAC treatment and that without NAC treatment (▲P < 0.01).
Mentions: HIF-1α protein levels were measured using Western blot after BSO pretreatment. When BSO concentration reached at 50 μM, the down-regulation of HIF-1α expression, under the hypoxia condition, was observed in HepG2 cells. It is then very clear that HIF-1α proteins in hypoxic cells were significantly decreased with BSO concentrations gradually increasing. In addition, the inhibition of HIF-1α expression was reversed by 5 mM NAC supplement. However, we also found that NAC failed to elevate the level of HIF-1α expression inhibited by BSO concentration at 200 μM. These results were shown in Figure 3

Bottom Line: To further confirm the effect of intracellular redox status on HIF-1α expression, N-acetylcysteine (NAC) was added to culture cells for 8 h before the hypoxia treatment.The results showed that BSO pretreatment down-regulated HIF-1α and the effect was concentration-dependent, on the other hand, the increases of intracellular GSH contents by NAC could partly elevate the levels of HIF-1α expression.The levels of P-Gp (MDR-1) and EPO were concomitant with the trend of HIF-1α expression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Teaching & Research Section of Nuclear Medicine, An-hui Medical University, Hefei, China. wensenjn@139.com

ABSTRACT
Hypoxia inducible factor-1 (HIF-1) has been considered as a critical transcriptional factor in response to hypoxia. It can increase P-glycoprotein (P-Gp) thus generating the resistant effect to chemotherapy. At present, the mechanism regulating HIF-1α is still not fully clear in hypoxic tumor cells. Intracellular redox status is closely correlated with hypoxic micro-environment, so we investigate whether alterations in the cellular redox status lead to the changes of HIF-1α expression. HepG2 cells were exposed to Buthionine sulphoximine (BSO) for 12 h prior to hypoxia treatment. The level of HIF-1α expression was measured by Western blot and immunocytochemistry assays. Reduce glutathione (GSH) concentrations in hypoxic cells were determined using glutathione reductase/5,5'-dithiobis-(2-nitrob-enzoic acid) (DTNB) recycling assay. To further confirm the effect of intracellular redox status on HIF-1α expression, N-acetylcysteine (NAC) was added to culture cells for 8 h before the hypoxia treatment. The levels of multidrug resistance gene-1 (MDR-1) and erythropoietin (EPO) mRNA targeted by HIF-1α in hypoxic cells were further determined with RT-PCR, and then the expression of P-Gp protein was observed by Western blotting. The results showed that BSO pretreatment down-regulated HIF-1α and the effect was concentration-dependent, on the other hand, the increases of intracellular GSH contents by NAC could partly elevate the levels of HIF-1α expression. The levels of P-Gp (MDR-1) and EPO were concomitant with the trend of HIF-1α expression. Therefore, our data indicate that the changes of redox status in hypoxic cells may regulate HIF-1α expression and provide valuable information on tumor chemotherapy.

Show MeSH
Related in: MedlinePlus