Chemical inhibitors destabilize HuR binding to the AU-rich element of TNF-alpha mRNA

Hu protein R (HuR) binds to the AU-rich element (ARE) in the 3'UTR to stabilize TNF-alpha mRNA. Here, we identified chemical inhibitors of the interaction between HuR and the ARE of TNF-alpha mRNA using RNA electrophoretic mobility gel shift assay (EMSA) and filter binding assay. Of 179 chemicals screened, we identified three with a half-maximal inhibitory concentration (IC(50)) below 10 micrometer. The IC(50) of quercetin, b-40, and b-41 were 1.4, 0.38, and 6.21 micrometer, respectively, for binding of HuR protein to TNF-alpha mRNA. Quercetin and b-40 did not inhibit binding of tristetraprolin to the ARE of TNF-alpha mRNA. When LPS-treated RAW264.7 cells were treated with quercetin and b-40, we observed decreased stability of TNF-alpha mRNA and decreased levels of secreted TNF-alpha. From these results, we could find inhibitors for the TNF-alpha mRNA stability, which might be used advantageously for both the study for post-transcriptional regulation and the discovery of new anti-inflammation drugs.

Depletion of mitochondrial DNA up-regulates the expression of MDR1 gene via an increase in mRNA stability

The mutation and reduction of mitochondrial DNA (mtDNA) have been suggested as factors in the carcinogenesis. However, whether the depletion of mtDNA induces multidrug resistance in cancer cells has not been fully investigated. To elucidate the association of cellular mtDNA content and drug resistance, we generated HCT-8 colon cancer cells which revealed a marked decrease in cellular mtDNA and ATP content, concomitant with a lack of mRNAs encoded by mtDNA. The mtDNA-depleted cells showed a decreased sensitivity and accumulation of anti-cancer drugs, suggesting that mtDNA depletion could develop multidrug resistance (MDR) phenotype in HCT-8 cells. We found that the expression level of MDR1 mRNA and its translated product P-glycoprotein was increased in the mtDNA- depleted cells, indicating that the decrease of sensitivity and accumulation of anti-cancer drug in the mtDNA-depleted cells might be due to a substantial increase in the expression of P-glycoprotein. Furthermore, increased expression of MDR1 mRNA and P-glycoprotein was due to an increase of mRNA stability rather than transcriptional activation. Taken together, these results indicate that mtDNA depletion can induce an increased P-glycoprotein expression via an increase of mRNA stability and suggest that the mtDNA depletion in cancer cells plays an important role in the induction of MDR phenotype.