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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 changes of redox status in hypoxic cells by different pretreatment. (A) showed the alteration of intracellular GSH and GSSG contents in HepG2 cells under hypoxic condition; (B) showed the ratios of GSH and GSSG in HepG2 cells under hypoxic condition. (◆p < 0.05, #p < 0.01, as compared with hypoxia control; ▲p < 0.05, *p < 0.01, as compared with the cells by NAC treatment).
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Figure 2: The changes of redox status in hypoxic cells by different pretreatment. (A) showed the alteration of intracellular GSH and GSSG contents in HepG2 cells under hypoxic condition; (B) showed the ratios of GSH and GSSG in HepG2 cells under hypoxic condition. (◆p < 0.05, #p < 0.01, as compared with hypoxia control; ▲p < 0.05, *p < 0.01, as compared with the cells by NAC treatment).

Mentions: As shown in Figure 2, BSO treatment led to significant reduction of intracellular GSH level and the effect was in a concentration-dependent manner. Intracellular GSSG contents were increased concomitant with BSO concentrations, resulting to subsequent reductions of GSH/GSSG ratios. The declines of GSH level were partially restored from hypoxic cells by the addition of 5 mM NAC prior to hypoxia. Compared with the cells in the absence of NAC, there was an increase in GSH/GSSG ratio in the presence of 5 mM NAC. It indicated that BSO inhibited the accumulation of GSH in cells, but the effect could be partially reversed by NAC treatment.


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 changes of redox status in hypoxic cells by different pretreatment. (A) showed the alteration of intracellular GSH and GSSG contents in HepG2 cells under hypoxic condition; (B) showed the ratios of GSH and GSSG in HepG2 cells under hypoxic condition. (◆p < 0.05, #p < 0.01, as compared with hypoxia control; ▲p < 0.05, *p < 0.01, as compared with the cells by NAC treatment).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: The changes of redox status in hypoxic cells by different pretreatment. (A) showed the alteration of intracellular GSH and GSSG contents in HepG2 cells under hypoxic condition; (B) showed the ratios of GSH and GSSG in HepG2 cells under hypoxic condition. (◆p < 0.05, #p < 0.01, as compared with hypoxia control; ▲p < 0.05, *p < 0.01, as compared with the cells by NAC treatment).
Mentions: As shown in Figure 2, BSO treatment led to significant reduction of intracellular GSH level and the effect was in a concentration-dependent manner. Intracellular GSSG contents were increased concomitant with BSO concentrations, resulting to subsequent reductions of GSH/GSSG ratios. The declines of GSH level were partially restored from hypoxic cells by the addition of 5 mM NAC prior to hypoxia. Compared with the cells in the absence of NAC, there was an increase in GSH/GSSG ratio in the presence of 5 mM NAC. It indicated that BSO inhibited the accumulation of GSH in cells, but the effect could be partially reversed by NAC treatment.

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