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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.

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The change of HIF-1α expression by ICC assay. (A) The picture of ICC was shown. a: negative control; b: normoxic control; c: hypoxic control; d: the hypoxic cells by 50 μM BSO pretreatment; e: the hypoxic cells by 100 μM BSO pretreatment; f: the hypoxic cells by 200 μM BSO pretreatment; g: the hypoxic cells by 50 μM BSO + 5 mM NAC pretreatment; j: the hypoxic cells by 100 μM BSO + 5 mM NAC pretreatment; k: the hypoxic cells by 200 μM BSO + 5 mM NAC pretreatment. (B) The results of statistical analysis were shown with H-score values of semi-quantitative evaluations. (◆P <0.05, #p < 0.01, compared with hypoxic control; *P <0.05, compared with the hypoxic cells by 5 mM NAC pretreatment).
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Figure 4: The change of HIF-1α expression by ICC assay. (A) The picture of ICC was shown. a: negative control; b: normoxic control; c: hypoxic control; d: the hypoxic cells by 50 μM BSO pretreatment; e: the hypoxic cells by 100 μM BSO pretreatment; f: the hypoxic cells by 200 μM BSO pretreatment; g: the hypoxic cells by 50 μM BSO + 5 mM NAC pretreatment; j: the hypoxic cells by 100 μM BSO + 5 mM NAC pretreatment; k: the hypoxic cells by 200 μM BSO + 5 mM NAC pretreatment. (B) The results of statistical analysis were shown with H-score values of semi-quantitative evaluations. (◆P <0.05, #p < 0.01, compared with hypoxic control; *P <0.05, compared with the hypoxic cells by 5 mM NAC pretreatment).

Mentions: To further verify the effect of redox status on HIF-1α levels, we detected the expressions of HIF-1α proteins by using immunocytochemistry technique (ICC). As shown in Figure 4, cells showed more negative staining than control group after BSO pretreatment and NAC decreased the inhibition. The results were basically consistent with Western blot result.


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 expression by ICC assay. (A) The picture of ICC was shown. a: negative control; b: normoxic control; c: hypoxic control; d: the hypoxic cells by 50 μM BSO pretreatment; e: the hypoxic cells by 100 μM BSO pretreatment; f: the hypoxic cells by 200 μM BSO pretreatment; g: the hypoxic cells by 50 μM BSO + 5 mM NAC pretreatment; j: the hypoxic cells by 100 μM BSO + 5 mM NAC pretreatment; k: the hypoxic cells by 200 μM BSO + 5 mM NAC pretreatment. (B) The results of statistical analysis were shown with H-score values of semi-quantitative evaluations. (◆P <0.05, #p < 0.01, compared with hypoxic control; *P <0.05, compared with the hypoxic cells by 5 mM NAC pretreatment).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: The change of HIF-1α expression by ICC assay. (A) The picture of ICC was shown. a: negative control; b: normoxic control; c: hypoxic control; d: the hypoxic cells by 50 μM BSO pretreatment; e: the hypoxic cells by 100 μM BSO pretreatment; f: the hypoxic cells by 200 μM BSO pretreatment; g: the hypoxic cells by 50 μM BSO + 5 mM NAC pretreatment; j: the hypoxic cells by 100 μM BSO + 5 mM NAC pretreatment; k: the hypoxic cells by 200 μM BSO + 5 mM NAC pretreatment. (B) The results of statistical analysis were shown with H-score values of semi-quantitative evaluations. (◆P <0.05, #p < 0.01, compared with hypoxic control; *P <0.05, compared with the hypoxic cells by 5 mM NAC pretreatment).
Mentions: To further verify the effect of redox status on HIF-1α levels, we detected the expressions of HIF-1α proteins by using immunocytochemistry technique (ICC). As shown in Figure 4, cells showed more negative staining than control group after BSO pretreatment and NAC decreased the inhibition. The results were basically consistent with Western blot result.

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