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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 MDR-1 expressions by RT-PCR and Western blotting measurement. Letter N means the cells under normoxic condition; Letter H means the cells under hypoxic condition: (A) The representative gel picture was taken from three separate RT-PCR experiments. (B) Compared with hypoxic control, the analysis of relative densities showed that there was statistical difference the experimental cells by 100 and 200 μM BSO pretreatment respectively (#p < 0.01). After NAC incubation, the expression of MDR-1 was elevated again, and there were significant difference between the group with 100 μM NAC treatment and that without NAC treatment (▲P < 0.05). (C) The representative gel picture was taken from three separate Western blotting experiments. (D) Compared with hypoxic control, the analysis of relative densities showed that there was statistical difference the experimental cells by 100 and 200 μM BSO pretreatment respectively (#p < 0.01). After NAC incubation, the expression of MDR-1 was elevated again, and there were significant difference between the group with 100 μM NAC treatment and that without NAC treatment (◆P < 0.01).
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Figure 5: The changes of MDR-1 expressions by RT-PCR and Western blotting measurement. Letter N means the cells under normoxic condition; Letter H means the cells under hypoxic condition: (A) The representative gel picture was taken from three separate RT-PCR experiments. (B) Compared with hypoxic control, the analysis of relative densities showed that there was statistical difference the experimental cells by 100 and 200 μM BSO pretreatment respectively (#p < 0.01). After NAC incubation, the expression of MDR-1 was elevated again, and there were significant difference between the group with 100 μM NAC treatment and that without NAC treatment (▲P < 0.05). (C) The representative gel picture was taken from three separate Western blotting experiments. (D) Compared with hypoxic control, the analysis of relative densities showed that there was statistical difference the experimental cells by 100 and 200 μM BSO pretreatment respectively (#p < 0.01). After NAC incubation, the expression of MDR-1 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: The levels of MDR-1 and EPO transcription were detected through semi-quantitative RT-PCR. The results displayed that the levels of MDR-1 and EPO mRNA were declined in hypoxic cells when BSO concentration was at 50 μM, but it wasn't shown that there was a statistical significance at the MDR-1 and EPO mRNA of 50 μM BSO pretreatment compared with those of the hypoxic control. Concomitant with the increases of BSO concentrations, the levels of MDR-1 and EPO mRNA in hypoxic cells were gradually decreased. And then the inhibitory effects on MDR-1 and EPO mRNA, BSO concentrations reaching at 100 μM and 200 μM respectively, were shown statistical differences. Meanwhile, NAC could reduce the inhibition of BSO to MDR-1 and EPO mRNA. Furthermore, the expression of P-gp by MDR-1 translation, tested with western blotting, was also confirmed with the change of MDR-1 mRNA. Above experimental results were displayed in Figure 5 and Figure 6. It is therefore clear that redox micro-environment may influence the levels of target genes located at the downstream of HIF-1.


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 MDR-1 expressions by RT-PCR and Western blotting measurement. Letter N means the cells under normoxic condition; Letter H means the cells under hypoxic condition: (A) The representative gel picture was taken from three separate RT-PCR experiments. (B) Compared with hypoxic control, the analysis of relative densities showed that there was statistical difference the experimental cells by 100 and 200 μM BSO pretreatment respectively (#p < 0.01). After NAC incubation, the expression of MDR-1 was elevated again, and there were significant difference between the group with 100 μM NAC treatment and that without NAC treatment (▲P < 0.05). (C) The representative gel picture was taken from three separate Western blotting experiments. (D) Compared with hypoxic control, the analysis of relative densities showed that there was statistical difference the experimental cells by 100 and 200 μM BSO pretreatment respectively (#p < 0.01). After NAC incubation, the expression of MDR-1 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 5: The changes of MDR-1 expressions by RT-PCR and Western blotting measurement. Letter N means the cells under normoxic condition; Letter H means the cells under hypoxic condition: (A) The representative gel picture was taken from three separate RT-PCR experiments. (B) Compared with hypoxic control, the analysis of relative densities showed that there was statistical difference the experimental cells by 100 and 200 μM BSO pretreatment respectively (#p < 0.01). After NAC incubation, the expression of MDR-1 was elevated again, and there were significant difference between the group with 100 μM NAC treatment and that without NAC treatment (▲P < 0.05). (C) The representative gel picture was taken from three separate Western blotting experiments. (D) Compared with hypoxic control, the analysis of relative densities showed that there was statistical difference the experimental cells by 100 and 200 μM BSO pretreatment respectively (#p < 0.01). After NAC incubation, the expression of MDR-1 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: The levels of MDR-1 and EPO transcription were detected through semi-quantitative RT-PCR. The results displayed that the levels of MDR-1 and EPO mRNA were declined in hypoxic cells when BSO concentration was at 50 μM, but it wasn't shown that there was a statistical significance at the MDR-1 and EPO mRNA of 50 μM BSO pretreatment compared with those of the hypoxic control. Concomitant with the increases of BSO concentrations, the levels of MDR-1 and EPO mRNA in hypoxic cells were gradually decreased. And then the inhibitory effects on MDR-1 and EPO mRNA, BSO concentrations reaching at 100 μM and 200 μM respectively, were shown statistical differences. Meanwhile, NAC could reduce the inhibition of BSO to MDR-1 and EPO mRNA. Furthermore, the expression of P-gp by MDR-1 translation, tested with western blotting, was also confirmed with the change of MDR-1 mRNA. Above experimental results were displayed in Figure 5 and Figure 6. It is therefore clear that redox micro-environment may influence the levels of target genes located at the downstream of HIF-1.

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