In this study, we have demonstrated the dark side of Nrf2. Since the discovery of Nrf2 and its important role in antioxidant response, Nrf2 has been reviewed as a ‘good’ transcription factor that is essential in protecting us from oxidative stress-related diseases. Many studies in the field have been focused on identification of Nrf2 activators to boost the Nrf2-dependent defense response for disease prevention. Here, we provide another scenario where activation of Nrf2 is ‘bad’ for cancer patients during the course of chemotherapy, as evidenced by our finding that modulation of the Nrf2-mediated response affects survival of cancer cells in response to chemotherapeutic agents. Stable overexpression of Nrf2, and therefore upregulation of the Nrf2-downstream effects, resulted in enhanced resistance of cancer cells to chemotherapeutic agents including cisplatin, doxorubicin and etoposide. Inversely, downregulation of the Nrf2 protein and its downstream response by overexpression of Keap1 or transient transfection of Nrf2–siRNA rendered cancer cells more susceptible to these drugs. In addition to genetic means of Nrf2 modulation, upregulation of Nrf2 by the small chemical tBHQ also enhanced the resistance of cancer cells, indicating the feasibility of using small chemical inhibitors of Nrf2 as adjuvants to chemotherapy to increase the efficacy of chemotherapeutic agents. Furthermore, this notion was tested in three different cancer cell lines such as lung carcinoma, breast adenocarcinoma and neuroblastoma with three different anticancer drugs, demonstrating that the strategy of using Nrf2 inhibitors to increase efficacy of chemotherapeutic agents is not specific to certain cancer types or anticancer drugs and thus can be applied during the course of chemotherapy to treat many cancer types. Based on these results, identification of small chemicals that potently and specifically inhibit the Nrf2-dependent response is extremely important and such chemicals can be used to increase the efficacy of anticancer drugs.
The data presented here were obtained using three cancer cell lines, neuroblastoma SH-SY5Y, breast adenocarcinoma MDA-MB-231 and non-small cell lung carcinoma A549. Except A549, in which Keap1 is mutated and underexpressed (28
), the status of Keap1 in MDA-MB-231 and SH-SY5Y has not been reported. We observed that the basal expression level of Nrf2 was high in A549, but low in MDA-MB-231 and SH-SY5Y (data not shown). Interestingly, stable overexpression of Keap1 or Nrf2–shRNA failed to knock down Nrf2 further in MDA-MB-231- and SH-SY5Y-derived cell lines, while stable overexpression of Nrf2 gave phenotypes as shown in and . Based on the report that Keap1 is mutated in some non-small cell lung cancers, along with our data showing the high Nrf2-downstream gene expression (NQO1 mRNA) in most stage II and stage III cancer tissues, it is possible that inhibition of Nrf2 expression during chemotherapy may be more effective in these tumors in which Keap1 is mutated/underexpressed and Nrf2 is upregulated. Further studies investigating the correlation between the status of Keap1 and the efficacy of Nrf2 inhibition on cancer cell sensitivity to chemotherapeutic agents are important. Herein, identification of potent Nrf2 inhibitors and investigation of Keap1 status in different tumors are two research directions to be focused on.
Upregulation of the Nrf2-mediated survival pathway indeed protects cancer cells from all three different chemotherapeutic agents tested. Etoposide kills cells by increasing the topoisomerase II-induced DNA fragmentation, which triggers programed cell death (32
). Similar to etoposide, doxorubicin is also classified as a topoisomerase II poison. However, doxorubicin exhibits a wide spectrum of cytotoxicity, possibly due to other mechanisms of action including generation of free radicals (33
). Cisplatin attacks DNA by forming cisplatin–DNA adducts that inhibit DNA replication and transcription (34
). Since Nrf2 regulates not only antioxidants but also a global cellular-defensive response, we reasoned that the Nrf2-dependent protection against chemotherapeutic agents should not be specific to ones that kill cancer cells by generation of reactive oxygen species. Although the number of anticancer drugs tested in this study is only limited to three, the sensitivity changes due to manipulation of the Nrf2 pathway were observed in response to all three drugs. These results indicate that the strategy of inhibiting Nrf2 during chemo treatment could be applied to broad range of anticancer drugs.
Our results clearly indicate that the Nrf2-dependent defense response helps survival of cancer cells during treatment with chemotherapeutic agents. It is unclear to what extent the Nrf2-dependent protection accounts for the drug resistance phenomena observed previously in many cancer types. Drug resistance during chemotherapy is the major obstacle to the successful treatment of many cancers including neuroblastoma, breast cancers and lung cancers. During chemotherapy, a strong initial response is frequently followed by the appearance of multidrug-resistant variants.
Several mechanisms are thought to account for the drug resistance phenotype. The multiple drug resistance-associated proteins have been reported to be responsible for decreased intracellular drug accumulation (35
). Increased levels of cellular thiols, facilitated detoxification of drugs and rapid DNA repair were found to be associated to drug resistance (34
). A recent report identified a function of p53 in determining multidrug sensitivity on neuroblastoma (38
). In addition, upregulation of activating transcription factor 4 (ATF4) was reported to increase cisplatin resistance in human cancer cell lines (39
). Interestingly, many genes reported to play roles in drug resistance seemingly have a functional link with Nrf2. For instance, Nrf2 was initially identified as a key regulator of phase II-detoxifying enzymes through antioxidant response elements in the promoter region of these genes. In addition, Nrf2 regulates many of the key enzymes that are important in maintaining cellular redox homeostasis, such as gamma-glutamylcysteine synthetase (γ-GCS), xCT cysteine/glutamate exchange transport and thioredoxin (40
). γ-GCS is a rate-limiting enzyme controlling glutathione biosynthesis, whereas xCT cysteine/glutamate exchange transport facilitates the uptake of cysteine that is needed for glutathione synthesis. Thioredoxin is another antioxidant protein that works in concert with glutathione to maintain intercellular redox homeostasis. In addition, many members of transporters including the multidrug resistance-associated protein 1-6 (MRP 1-6), have been reported as Nrf2 target genes (45
). ATF4 was reported to upregulate HO-1 transcription by forming heterodimer with Nrf2 (48
). p53 and Nrf2 were found to work cooperatively in protecting against N
-nitrosobutyl (4-hydroxybutyl) amine-induced bladder cancer (49
). Collectively, these results implicate that upregulation of Nrf2 is responsible, at least in part, for drug resistance observed during the course of chemotherapy.
Consistent with the notion that Nrf2 protects cancer cells from killing by anticancer drugs, a recent paper reported that Nrf2-knockout murine embryonic fibroblasts were more sensitive to cisplatin treatment (50
). Transient knockdown of Nrf2 with Nrf2–siRNA decreased survival of human ovarian cancer cells under cisplatin treatment (50
). In analog to Nrf2, expression of HO-1 in many tumor tissues was also upregulated (51
). Furthermore, it has been shown that decreased expression of HO-1 by either chemical inhibitor or HO-1 siRNA sensitized cells to cytotoxicity of cisplatin, whereas upregulation of HO-1 attenuates the toxicity induced by cisplatin or by photodynamic therapy (53
). Since Nrf2 regulates many genes including HO-1, targeting Nrf2 may be more effective than targeting HO-1. In conclusion, our data demonstrate that inhibition of Nrf2 sensitizes cells to chemotherapeutic drugs, suggesting that Nrf2 inhibitors may be used concomitantly to increase the efficacy of anticancer therapy.
Identification of the dark side of Nrf2, that is upregulation of Nrf2 in cancer cells provides them with a growth advantage under detrimental environments, may generate a concern in using Nrf2 activators for the purpose of chemoprevention. However, it seems worth mention that induction of the Nrf2-dependent defensive response by Nrf2 activators in normal tissue is transient because the negative regulator Keap1 is only inhibited temporarily and partially. However, in cancer tissues, dysregulation of Nrf2 by Keap1 due to mutation of Keap1 results in strong and persistent induction of Nrf2. Previously, it was shown that induction of Nrf2 by tBHQ reached a peak following 18–24 h treatment and was gradually diminished after 24 h (57). Thus, it would probably be safe to use Nrf2-activating chemopreventive drugs every other day. In the case of cancer patients undergoing treatment with chemotherapeutic drugs, it will be wise to temporally inhibit the Nrf2-dependent cytoprotection using Nrf2 inhibitors to enhance patients’ response to anticancer drugs. In the present work, we provide the strong evidence that response of cancer cells to chemotherapeutic drugs can be elevated by inhibiting the Nrf2-dependent pathway. Identification of small chemicals that specifically inhibit Nrf2, along with animal studies and human clinical trials, is needed to test the efficacy of coadministration of Nrf2 inhibitors during chemotherapy.