Identification of aldo-keto reductases as NRF2-target marker genes in human cells.

Transcription factor NF-E2-related factor 2 (NRF2) plays a crucial role in the cellular defense against oxidative/electrophilic stress by up-regulating multiple antioxidant genes. Numerous studies with genetically modified animals have demonstrated that Nrf2 is a sensitivity determining factor upon the exposure to environmental chemicals including carcinogens. Moreover, recent studies have demonstrated that polymorphism in the human NRF2 promoter is associated with higher risks for developing acute lung injury, gastric mucosal inflammation, and nephritis. Therefore, the identification of reliable and effective human target genes of NRF2 may allow the monitoring of NRF2 activity and to predict individual sensitivity to environmental stress-induced damage. For this purpose, we investigated genes that are tightly controlled by NRF2 to establish markers for NRF2 activity in human cells. Firstly, in the normal human renal epithelial HK-2 cells, the measurement of the expression of 30 previously reported NRF2 target genes in response to NRF2 inducers (sulforaphane, tert-butylhydroquinone, cinnamic aldehyde, and hydrogen peroxide) showed that the aldo-keto reductase (AKR) 1C1 is highly inducible by all treatments. Accordantly, the basal and inducible expressions of AKRs were significantly attenuated in NRF2-silenced HK-2 cells. Whereas, cells with stable KEAP1 knockdown, which causes a modest NRF2 activation, demonstrated substantially increased levels of AKR1A1, 1B1, 1B10, 1C1, 1C2, and 1C3. Secondly, the linkage between NRF2 and the AKRs was confirmed in human monocytic leukemia cell line U937, which can be a model of peripherally available blood cells. The treatment of U937 cells with NRF2 inducers including sulforaphane effectively elevated the expression of AKR1B1, 1B10, 1C1, 1C2, and 1C3. Whereas, the levels of both the basal and sulforaphane-inducible expression of AKR1C1 were significantly reduced in NRF2-silenced stable U937 cells compared to the control cells. Similarly, the inducible expression of AKR1C1 was observed in another human monocytic leukemia cell line THP-1 as well as in human primary blood CD14(+) monocytes. In conclusion, together with the high inducibility and NRF2 dependency shown in renal epithelial cells as well as in peripherally available blood cells, current findings suggest that AKRs can be utilized as a marker of NRF2 activity in human cells.

Cell growth inhibition and apoptosis by SDS-solubilized single-walled carbon nanotubes in normal rat kidney epithelial cells.

Carbon nanotubes (CNTs), promising novel nanomaterials, have been applied to drug delivery and bio-imaging; however, their potential harmful effects on human health and environment have gained much attention recently. In the present study, we investigated cytotoxic effect of solubilized single-walled CNTs (SWCNTs), which were dispersed in water by sodium dodesyl sulfate (SDS), in normal rat kidney epithelial cells (NRK-52E). SDS-SWCNT (0.125-10 µg/mL)-treated NRK-52E cells showed decreased cell viability and enhanced cytotoxicity marker levels following 24-48 h incubation. In addition, SDS-SWCNT treatment evoked the cell growth inhibition: 8 µg/mL SDS-SWCNT induced the growth arrest at G(0)/G(1) phase and levels of cell cycle-related proteins such as CDK2, CDK6 and phosphorylated-retinoblastoma (pRB) were significantly reduced by CNT. Whereas, at higher concentration of SDS-SWCNT, the percentage of cell numbers in apoptotic sub-G(1) phase was substantially increased. Along with these changes, SDS-SWCNT treatment elevated protein levels for p53 and p21 with a concomitant increase in the single strand DNA breakage. Taken together, these results suggest that SDS-solubilized SWCNTs exert genotoxic effect in renal epithelial cells, and p53-dependent signaling can be associated with the growth arrest and apoptosis events upon CNT-induced DNA damage.

Transcription factor Nrf2 maintains the basal expression of Mdm2: An implication of the regulation of p53 signaling by Nrf2.

Co-operated regulation of oxidative stress-response transcription factors would be an important issue for animals to determine the cell fate under environmental stress. This notion raises a possibility that NF-E2-related factor 2 (Nrf2), which confers cytoprotection against oxidative stress, and p53 can have a direct co-regulation network. In the current study, we have indentified that the expression of murine double minute 2 (Mdm2) is repressed in nrf2-deleted murine embryonic fibroblasts (MEFs). This was confirmed by microarray, RT-PCR, and immunoblot analyses, and further promoter analysis showed that Nrf2 is directly involved in the basal expression of Mdm2 through the antioxidant response element, which is located in the first intron of this gene. This linkage between Nrf2 and Mdm2 appears to cause the accumulation of p53 protein in nrf2-deficent MEFs. In addition, we show that ovarian carcinoma A2780 cells with Nrf2 shRNA expression displayed higher levels of p53 activation in response to hydrogen peroxide treatment, leading to increased cell death. Collectively, our results suggest novel evidence that the inhibition of Nrf2 can suppress Mdm2 expression, which may result in p53 signaling modulation. In addition, this observation supports the concept that Nrf2 inhibition in cancer cells can facilitate apoptotic response upon environmental stress.

G(alpha)12/13 inhibition enhances the anticancer effect of bortezomib through PSMB5 downregulation.

Bortezomib is a proteasome inhibitor approved for anticancer therapy. However, variable sensitivity of tumor cells exists in this therapy probably due to differences in the expression of proteasome subunits. G(alpha)(12/13) serves modulators or signal transducers in diverse pathways. This study investigated whether cancer cells display differential sensitivity to bortezomib with reference to G(alpha)(12/13) expression, and if so, whether G(alpha)(12/13) affects the expression of proteasome subunits and their activities. Bortezomib treatment exhibited greater sensitivities in Huh7 and SNU886 cells (epithelial type) than SK-Hep1 and SNU449 cells (mesenchymal type) that exhibited higher levels of G(alpha)(12/13). Overexpression of an active mutant of G(alpha)(12) (Galpha(12)QL) or G(alpha)(13) (G(alpha)(13)QL) diminished the ability of bortezomib to induce cytotoxicity in Huh7 cells. Moreover, transfection with the minigene that disturbs G protein-coupled receptor-G protein coupling (CT12 or CT13) increased it in SK-Hep1 cells. Consistently, MiaPaCa2 cells transfected with CT12 or CT13 exhibited a greater sensitivity to bortezomib. Evidence of G(alpha)(12/13)'s antagonism on the anticancer effect of bortezomib was verified in the reversal by G(alpha)(12)QL or G(alpha)(13)QL of the minigenes' enhancement of cytotoxity. Real-time polymerase chain reaction assay enabled us to identify PSMB5, multicatalytic endopeptidase complex-like-1, and proteasome activator subunit-1 repression by CT12 or CT13. Furthermore, G(alpha)(12/13) inhibition enhanced the ability of bortezomib to repress PSMB5, as shown by immunoblotting and proteasome activity assay. Moreover, this inhibitory effect on PSMB5 was attenuated by G(alpha)G(alpha)(12)QL or G(alpha)(13)QL. In conclusion, the inhibition of G(alpha)(12/13) activities may enhance the anticancer effect of bortezomib through PSMB5 repression, providing insight into the G(alpha)(12/13) pathway for the regulation of proteasomal activity.