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Flumethasone enhances the efficacy of chemotherapeutic drugs in lung cancer by inhibiting Nrf2 signaling pathway
Journal Pre-proof Flumethasone enhances the efficacy of chemotherapeutic drugs in lung cancer by inhibiting Nrf2 signaling pathway Yunjiang Zhou, Yang Zhou, Keke Wang, Tao Li, Mengdi Yang, Rui Wang, Yaxin Chen, Mengran Cao, Rong Hu PII:
S0304-3835(20)30018-5
DOI:
https://doi.org/10.1016/j.canlet.2020.01.010
Reference:
CAN 114652
To appear in:
Cancer Letters
Received Date: 23 September 2019 Revised Date:
9 January 2020
Accepted Date: 13 January 2020
Please cite this article as: Y. Zhou, Y. Zhou, K. Wang, T. Li, M. Yang, R. Wang, Y. Chen, M. Cao, R. Hu, Flumethasone enhances the efficacy of chemotherapeutic drugs in lung cancer by inhibiting Nrf2 signaling pathway, Cancer Letters, https://doi.org/10.1016/j.canlet.2020.01.010. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.
Abstract Nuclear factor erythroid-2-related factor 2 (Nrf2), a transcription factor, participates in protecting cells from electrophilic or oxidative stresses through regulating expression of cytoprotective and antioxidant genes. It has become one of the emerging targets for cancer chemosensitization, and small molecule inhibitors of Nrf2 can enhance the efficacy of chemotherapeutic drugs. Here, we found that flumethasone, a glucocorticoid, inhibited Nrf2 signaling in A549 and H460 cells by promoting Nrf2 protein degradation. Moreover, flumethasone significantly increased the sensitivity of A549 and H460 cells to chemotherapeutic drugs including cisplatin, doxorubicin and 5-FU. In mice bearing A549-shControl cells-derived xenografts, the size and weight of xenografts in the flumethasone and cisplatin combination group had a significant reduction compared with those in the cisplatin group, while in mice bearing A549-shNrf2 cells-derived xenografts, the size and weight of the xenografts in the combination group had no significant difference compared with those in the cisplatin group, demonstrating that chemosensitization effect of flumethasone is Nrf2-dependent. This work suggests that flumethasone can potentially be used as an adjuvant sensitizer to enhance the efficacy of chemotherapeutic drugs in lung cancer.
Keywords: Flumethasone, Nrf2, lung cancer, chemosensitization
Flumethasone enhances the efficacy of chemotherapeutic drugs in lung cancer by inhibiting Nrf2 signaling pathway Yunjiang Zhoua, Yang Zhoua, Keke Wanga, Tao Lia, Mengdi Yanga, Rui Wanga, Yaxin Chena, Mengran Caoa, Rong Hua, *
a
State Key Laboratory of Natural Medicines, School of Basic Medicine and
Clinical Pharmacy, China Pharmaceutical University, Nanjing, Jiangsu, China
* To whom correspondence should be addressed. School of Basic Medicine and Clinical Pharmacy, China Pharmaceutical University, 24 Tongjia Xiang, Nanjing, Jiangsu 210009, China. Email: [email protected] (Rong Hu).
Abstract Nuclear factor erythroid-2-related factor 2 (Nrf2), a transcription factor, participates in protecting cells from electrophilic or oxidative stresses through regulating expression of cytoprotective and antioxidant genes. It has become one of the emerging targets for cancer chemosensitization, and small molecule inhibitors of Nrf2 can enhance the efficacy of chemotherapeutic drugs. Here, we found that flumethasone, a glucocorticoid, inhibited Nrf2 signaling in A549 and H460 cells by promoting Nrf2 protein degradation. Moreover, flumethasone significantly increased the sensitivity of A549 and H460 cells to chemotherapeutic drugs including cisplatin, doxorubicin and 5-FU. In mice bearing A549-shControl cells-derived xenografts, the size and weight of xenografts in the flumethasone and cisplatin combination group had a significant reduction compared with those in the cisplatin group, while in mice bearing A549-shNrf2 cells-derived xenografts, the size and weight of the xenografts in the combination group had no significant difference compared with those in the cisplatin group, demonstrating that chemosensitization effect of flumethasone is Nrf2-dependent. This work suggests that flumethasone can potentially be used as an adjuvant sensitizer to enhance the efficacy of chemotherapeutic drugs in lung cancer.
Keywords: Flumethasone, Nrf2, lung cancer, chemosensitization
Abbreviations: Nrf2, nuclear factor erythroid 2-related factor 2; Keap1, Kelch-like ECH-associated protein 1; ARE, antioxidant response element; FLM, flumethasone; NQO1, NAD(P)H quinone oxidoreductase 1; HO-1, heme oxygenase-1; mRNA, messenger RNA; shRNA, short hairpin RNA; qRT-PCR, quantitative reverse transcription-PCR; IHC, immunohistochemistry; 8-OHdG, 8-hydroxydeoxyguanosine; GCR, glucocorticoid receptor; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen
1. Introduction Lung cancer is one of the most common malignant tumors and the leading cause of cancer-related death in the world [1]. Lung cancer is generally hard to be detected in the early stage, and therefore, patients are often diagnosed at mid or late stages. Although many chemotherapeutic drugs have been developed to treat lung cancer, the death rate is still rising. The main reason is that patients often develop chemoresistance during the course of chemotherapy. Therefore, it is urgent to explore the mechanism of chemoresistance and search for safe and effective chemotherapeutic sensitizers to increase the efficacy of chemotherapeutic drugs. Nrf2 participates in protecting cells from electrophilic or oxidative stresses by up-regulating expression of cytoprotective and antioxidant genes [2, 3]. Under normal conditions, Nrf2 is located in the cytoplasm and binds to the Kelch domain of Kelch-like ECH-associated protein 1 (Keap1) through the ETGE and DLG motifs of its Neh2 domain. Meanwhile, binding of Keap1 to Cul3-Rbx1-E3 ligase results in ubiquitination of Nrf2, which leads to proteasomal degradation of Nrf2 protein [4-6]. Therefore, Nrf2 protein is usually maintained at a low level in the cytoplasm. Under oxidative or electrophilic pressure, modification of Keap1 at cysteine residues blocks its interaction with Nrf2, leading to reducing Keap1-mediated degradation of Nrf2 [7]. Subsequently, Nrf2 accumulates in the cytoplasm and translocates into the nucleus, where it forms heterodimer with sMaf protein, which then binds to antioxidant response element (ARE) in the promoter region of Nrf2 target genes to promote their transcription [2, 8, 9]. Recent studies have shown that Nrf2 is overexpressed in a variety of cancers [10-13]. Meanwhile, high mutation rate of Keap1 is found in tumor tissues of lung cancer patients and such mutations lead to loss of Keap1 function and activate the Nrf2
signaling,
which
promote
cancer
progression,
chemoresistance
and
radioresistance [14-18]. Moreover, silencing Nrf2 or overexpressing Keap1 can increase the efficacy of chemotherapeutic drugs, and overexpression of Nrf2 will reduce it [19-22]. Therefore, Nrf2 may be a promising target for enhancing the
efficacy of chemotherapeutic drugs and combination of Nrf2 inhibitors with chemotherapeutic drugs may be an effective treatment for cancers. In this study, we identified flumethasone (FLM), a glucocorticoid with anti-inflammatory, vasoconstrictive and anti-hyperplasia properties, as a novel Nrf2 inhibitor. Furthermore, flumethasone could enhance the sensitivity of lung cancer to chemotherapeutic drugs. Therefore, flumethasone may be a promising agent to enhance the efficacy of chemotherapeutic drugs in lung cancer.
2. Materials and methods 2.1. Materials See supplementary materials for more details.
2.2. Cell culture and MTT assay All experimental procedures were performed using a standard protocol. See supplementary materials and methods for detailed experimental procedures.
2.3. Western blot assay, immunofluorescence assay, quantitative real-time PCR assay, CHX-chase analysis and ubiquitination assay All experimental procedures were performed using a standard protocol. See supplementary materials and methods for detailed experimental procedures.
2.4. Transfection of Nrf2 shRNA and Nrf2 plasmid All experimental procedures were performed using a standard protocol. See supplementary materials and methods for detailed experimental procedures.
2.5. Animal treatment Female BALB/c nude mice (18 ± 2 g, 6 weeks old) were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd (Beijing, China). Animal experiments were approved by the Animal Ethics Committee of China Pharmaceutical
University (Ethic approval number: 2019-11-003) and carried out following the guidelines in the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. See supplementary materials and methods for detailed animal experimental procedures.
2.6.
Immunohistochemistry
(IHC),
HE
staining
and
determination
of
8-hydroxydeoxyguanosine (8-OHdG) All experimental procedures were performed using a standard protocol. See supplementary materials and methods for detailed experimental procedures.
2.7. Human lung cancer tissue microarray A human lung cancer tissues microarray was purchased from Shanghai Outdo Biotech (Shanghai, China), which contains 94 cases of non-small-cell lung cancer tissues, with 86 cases having adjacent tissues. All patients had been pathologically diagnosed with non-small-cell lung cancer. IHC staining was used to analyze the expression of Nrf2 in tissues.
2.8. Statistical analysis The χ2 test was used to analyze the association between Nrf2 expression and clinicopathologic variables and Kaplan-Meier analysis was used to analyze the relationship between Nrf2 expression and patient survival. The other results were expressed as mean ± SD and were representative of three independent experiments. Statistical analysis was performed with the t-test for two groups or one-way ANOVA for multiple groups by using SPSS statistical software. P<0.05 was considered significant.
3. Results 3.1. Nrf2 is overexpressed in primary lung cancer samples and predicts poor prognosis
IHC staining was used to detect Nrf2 protein levels in lung cancer tissues microarray, and it was found that Nrf2 protein levels were higher in cancer tissues when compared with those in adjacent tissues (Fig. 1A). Moreover, about 67.4% of lung cancer patients had higher Nrf2 protein level in tumor tissues than in adjacent tissues (Fig. 1B). Among all lung cancer tissues in this tissue array, 64.9% had high Nrf2 expression (IHC scores 2 or 3, Fig. 1D), while in all adjacent tissues, 2.3% had high Nrf2 expression (IHC scores 2 or 3, Fig. 1D). In addition, the correlation between Nrf2 expression and patients’ survival was analyzed and it was found that lung cancer patients with high Nrf2 expression showed poor prognosis (Fig. 1E). Moreover, Nrf2 expression was correlated with the clinicopathological characteristics such as tumor size, clinical stage, T status and N status (Table 1). These results suggest that Nrf2 is overexpressed in primary lung cancer tissues and predicts poor prognosis. Next, the protein levels of Nrf2 in human lung cancer cells (A549, H460 and H1299) and normal bronchial epithelial cells (BEAS-2B and 16HBE) were detected by using western blot and it was found that Nrf2 protein levels in lung cancer cells were markedly higher than those in normal bronchial epithelial cells (Fig. 1F). Moreover, the mRNA levels of Nrf2 target genes in lung cancer cells were higher than those in normal bronchial epithelial cells (Fig. 1G). These data demonstrate that Nrf2 signaling is activated in lung cancer.
3.2. The expression of Nrf2 is associated with sensitivity of lung cancer cells to chemotherapeutic drugs To investigate whether Nrf2 expression is associated with the efficacy of chemotherapeutic drugs, Nrf2 shRNAs (shNrf2 #1 and shNrf2 #2) and Nrf2 overexpression plasmids were used to silence and overexpress Nrf2 in A549 and H460 cells respectively. We found that Nrf2 shRNAs decreased and Nrf2 overexpression plasmid increased the expression of Nrf2 in A549 and H460 cells (Fig. 2A, C, E, G and Supplementary Fig. S1). Moreover, the silencing efficiency of shNrf2 #2 was slightly stronger than that of shNrf2 #1 (Supplementary Fig. S1). Therefore, shNrf2 #2 was used to silence Nrf2 in the subsequent experiments. MTT assay was
then performed to detect the cytotoxicity of chemotherapeutic drugs including cisplatin, doxorubicin and 5-FU in A549 and H460 cells with Nrf2 knockdown or overexpression. The results showed that the sensitivity of A549 and H460 cells with Nrf2 knockdown to chemotherapeutic drugs was significantly increased (Fig. 2B, D), while the sensitivity of A549 and H460 with Nrf2 overexpression was significantly decreased (Fig. 2F, H), suggesting that Nrf2 expression is reversely correlated with the efficacy of chemotherapeutic drugs.
3.3. Flumethasone inhibits Nrf2 protein levels in A549 and H460 cells The inhibition on Nrf2 protein expression by flumethasone was then detected in A549 and H460 cells. As shown in Fig. 3A-B, 50, 100 and 200 nM of flumethasone significantly inhibited Nrf2 protein levels in cells. Moreover, it was found that Nrf2 protein levels in cells were significantly reduced at 6, 12 and 24 h after 100 nM of flumethasone treatment (Fig. 3C-D). In addition, endogenous Nrf2 immunostaining further confirmed that flumethasone treatment reduced the Nrf2 protein levels in A549 and H460 cells (Fig. 3E-F).
3.4. Flumethasone reduces Nrf2 protein levels by promoting Nrf2 protein degradation in A549 and H460 cells. The mRNA levels of Nrf2 and Keap1 in A549 and H460 cells after flumethasone treatment were detected by qRT-PCR, and the results showed that there was no significant difference in the mRNA levels of Nrf2 and Keap1 between flumethasone treated cells vs. control (Fig. 4A-D), suggesting that flumethasone does not inhibit Nrf2 at the transcription level. Therefore, we investigated whether flumethasone reduces Nrf2 protein level by promoting Nrf2 degradation. As shown in Fig. 4E-F, the treatment of MG132 (a proteasomal inhibitor) blocked the inhibitory effect of flumethasone on Nrf2 expression. Moreover, we found that flumethasone could significantly shorten the degradation half-life of Nrf2 (Fig. 4G-H), while ubiquitination analysis showed that ubiquitination levels of Nrf2 were significantly increased in cells after treatment with flumethasone (Fig. 4I-J). In the meantime,
ubiquitination levels of Keap1 in cells did no change significantly after treatment with flumethasone (Fig. 4I-J), indicating that flumethasone-induced degradation of Nrf2 protein is Keap1-independent. These results suggest that flumethasone reduces Nrf2 protein levels by promoting Nrf2 protein degradation in A549 and H460 cells. To investigate the role of glucocorticoid receptor (GCR) in flumethasone-induced down-regulation of Nrf2, two specific glucocorticoid receptor shRNAs (shGCR #1 and shGCR #2) were used to silence GCR expression in A549 and H460 cells. It was found that GCR knockdown blocked the inhibitory effects of flumethasone on Nrf2 (Supplementary Fig. S2A-B), indicating that flumethasone-induced down-regulation of Nrf2 is GCR-dependent. We also examined the effect of flumethasone on ubiquitination of Nrf2 in A549 and H460 cells with GCR knockdown and found that flumethasone could not significantly promote ubiquitination of Nrf2 in A549 and H460 cells with GCR knockdown (Supplementary Fig. S2C-D), indicating that flumethasone-mediated Nrf2 protein degradation is GCR-dependent.
3.5. Flumethasone sensitizes lung cancer cells with high Nrf2 expression to chemotherapeutic drugs Next, we investigated the inhibition on Nrf2 protein and its target genes expression by flumethasone treatment in A549, H460, H1299, BEAS-2B and 16HBE cells, and found that flumethasone significantly inhibited the protein levels of Nrf2 and its target genes in A549 and H460 cells, with a weaker inhibition in H1299 cells, and no effect in BEAS-2B and 16HBE cells (Fig. 5A-B). When flumethasone was used in combination with cisplatin, doxorubicin or 5-FU in A549, H460, H1299, BEAS-2B and 16HBE cells, it had strong chemosensitization effects in A549 and H460 cells, which highly express Nrf2, and relatively weak chemosensitization effects in H1299, BEAS-2B and 16HBE cells, with lower Nrf2 levels (Fig. 5C-E and Supplementary Table S2-4). These results demonstrate that flumethasone enhances sensitivity of lung cancer cells to chemotherapeutic drugs in those with relatively high Nrf2 levels.
3.6. Flumethasone enhances the sensitivity of A549 and H460 cells to chemotherapeutic drugs by inhibiting Nrf2 signaling To
investigate
whether
flumethasone-induced
chemosensitization
is
Nrf2-dependent, Nrf2 shRNA was used to silence Nrf2 expression in A549 and H460 cells, and then the cytotoxicity of chemotherapeutic drugs including cisplatin, doxorubicin and 5-FU in these cells were measured. When A549-shNrf2 and H460-shNrf2 cells were treated with 100 nM of flumethasone, Nrf2, NQO1 and HO-1 protein levels did not change significantly (Fig. 6A, C), and flumethasone could not increase the sensitivity of A549-shNrf2 and H460-shNrf2 cells to chemotherapeutic drugs (Fig. 6B, D). These data suggest that chemosensitization effect of flumethasone is dependent on its down-regulation of Nrf2 signaling. To further confirm this, Nrf2 overexpression plasmids were used to restore Nrf2 protein levels in cells (Fig. 6E, G), and it could eliminate flumethasone-induced chemosensitization in cells (Fig. 6F, H). These results further demonstrate that flumethasone-induced chemosensitization is Nrf2-dependent.
3.7. Flumethasone enhances the sensitivity of A549 cells-derived xenografts to cisplatin by inhibiting Nrf2 signaling To
investigate
whether
flumethasone-induced
chemosensitization
was
Nrf2-dependent in vivo, we constructed A549-shControl and A549-shNrf2 cells-derived xenografts mouse models. In A549-shControl cells-derived xenograft mice, the size and weight of xenografts in the flumethasone and cisplatin combination group had a significant reduction compared with those in the cisplatin group (Fig. 7A). However, in A549-shNrf2 cells-derived xenograft mice, the size and weight of the xenografts in the combination group had no significantly difference compared with those in the cisplatin group (Fig. 7B). Moreover, flumethasone could significantly inhibit Nrf2, NQO1 and HO-1 protein levels in A549-shControl cells-derived xenografts, while no inhibition was observed in A549-shNrf2 cells-derived xenografts (Fig. 7C-E). In addition, the levels of 8-OHdG and cleaved caspase-3 were also detected in tumor tissues and it was found that combination of flumethasone with
cisplatin significantly increased the levels of 8-OHdG and cleaved caspase-3 (Supplementary Fig. S3), indicating that flumethasone promotes cisplatin-induced apoptosis by increasing redox status. These results suggest that flumethasone enhances the sensitivity of A549 cells-derived xenografts to cisplatin by inhibiting Nrf2 signaling. We also investigated the toxicity of flumethasone and cisplatin alone or in combination in these mice. As shown in Fig. 7A-B, body weight of the mice did not change significantly during the experiment. Moreover, the pathological morphology and organ indexes of mice in the vehicle group, flumethasone group, cisplatin group and combination group had no significant difference (Fig. 8A-B). In addition, we examined the plasma levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and blood urea nitrogen (BUN) and found that there is no significant difference among all groups (Fig. 8C-E). Therefore, toxicity was not observed in mice at the concentrations used here in our study.
4. Discussion Chemoresistance is the major obstacle to successful treatment of cancers. Recent studies have demonstrated that overactivation of Nrf2 can lead to chemoresistance in lung cancer as well as other types of cancers [21, 23, 24]. By using Nrf2 siRNA to inhibit Nrf2 expression in cancer cells, researchers found that the growth of cancer cells was slowed and the sensitivity of cancer cells to chemotherapeutic drugs was increased [21]. Consistent with previous studies, we found that Nrf2 was highly expressed in tumor tissues of lung cancer patients and silencing Nrf2 in lung cancer cells could increase the sensitivity of these cells to chemotherapeutic drugs. Moreover, we also found that the expression of Nrf2 in tumor tissues of lung cancer patients was associated with prognosis and clinicopathological characteristics. These results indicate that Nrf2 may be an effective target in the treatment of lung cancer, and therefore, combination of Nrf2 inhibitors with chemotherapeutic drugs may be an useful strategy for the treatment of lung cancer.
In previous studies, small-molecule Nrf2 inhibitors, such as epigallocatechin 3-gallate [25], luteolin [26], alkaloid trigonelline [27], brusatol [28], halofuginone [29] and camptothecin [30], have been shown to increase chemosensitivity of cancers to chemotherapeutic drugs, indicating that they can potentially be used as chemotherapeutic sensitizers. We have previously reported that wogonin reverses doxorubicin resistance of MCF-7/DOX cells through suppressing Nrf2 signaling [31]. However, its clinical side effects are unclear. We also found that digoxin can reverse gemcitabine resistance in pancreatic cancer cells through inhibiting Nrf2 signaling [32], while long-term use of digoxin might cause cardiotoxicity in humans. In the present study, flumethasone was found to inhibit the activity of Nrf2 at nanomolar concentrations, and it sensitized lung cancer cells with high Nrf2 expression to chemotherapeutic drugs. Moreover, flumethasone-mediated chemosensitization of lung cancer relied on its ability to inhibit the Nrf2 signaling pathway. In our in vivo experiments, flumethasone and cisplatin in combination did not significantly change the body weight, organ indexes and pathological morphology in mice, indicating that combination of flumethasone and cisplatin at the doses used here does not have obvious toxicity. Therefore, flumethasone may be used as an adjuvant in lung cancer treatment. Both the transcription process of genes and the degradation process of proteins will affect the protein level in cells. In this study, we found that flumethasone treatment did not change Nrf2 mRNA levels in A549 and H460 cells, indicating that flumethasone does not regulate Nrf2 at transcription level. We then investigated whether flumethasone regulates Nrf2 protein degradation and found that flumethasone could decrease Nrf2 protein levels by promoting its degradation. It should be noted that Keap1 mutations were found in A549 and H460 cells, which led to the lack of interaction of Keap1-Nrf2 in these cells [14]. Therefore, Keap1-dependent Nrf2 protein degradation should be minimal in these cells, and we speculated that flumethasone-mediated
Nrf2
degradation
in
A549
and
H460
cells
are
Keap1-independent. Indeed, we found that flumethasone did not increase ubiquitination
of
Keap1
in
A549
and
H460
cells,
suggesting
that
flumethasone-mediated Nrf2 degradation is Keap1-independent. Previously reports showed that glucocorticoids inhibited Nrf2 signaling through the activation of GCR and potentiated the antitumor effects of chemotherapeutic drugs [33-35]. Here, we found
that
flumethasone-induced
down-regulation
of
Nrf2
protein
was
GCR-dependent. It has been reported that β-TrCP and Hrd1 are involved in regulating the degradation of Nrf2 in a Keap1-independent manner under certain circumstances [36-38]. However, it is not clear whether β-TrCP and Hrd1 are also involved in the flumethasone-mediated regulation of Nrf2 degradation in A549 and H460 cells. In summary, this study demonstrates that flumethasone inhibits Nrf2 signaling pathway through promoting degradation of Nrf2 protein and enhances the efficacy of chemotherapeutic drugs in lung cancer cells (Fig. 9). We also provided in vitro and in vivo evidences that flumethasone in combination with cisplatin could be a potential therapeutic strategy in treating lung cancer which highly express Nrf2.
Acknowledgements This work was supported by the National Natural Science Foundation of China (No.81372268, No.81672816 and No.81872337), the Program for Jiangsu Province Innovative Research (KYLX16_1120), the Natural Science Foundation for Distinguished Young Scholars of Jiangsu Province (No. BK20130026).
Conflict of interest There is no conflict of interest.
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Table 1 Association between clinicopathologic variables of lung cancer patients and Nrf2 expression. Variables
Group
Sex Age Tumor size Stage
T status
N status
M status
Nrf2 expression
Total
P-value
35 (66%)
53
0.792
15 (36.6%)
26 (63.4%)
41
≤ 60 years
18 (36.7%)
31 (63.3%)
49
> 60 years
15 (33.3%)
30 (66.7%)
45
≤ 3 cm
17 (50%)
17 (50%)
34
> 3 cm
16 (26.7%)
44 (73.3%)
60
Ⅰ
17 (56.7%)
13 (43.3%)
30
Ⅰ-Ⅰ
16 (25.4%)
47 (74.6%)
63
Missing
0 (0%)
1 (100%)
1
T1
14 (70%)
6 (30%)
20
T2
14 (28%)
36 (72%)
50
T3
5 (27.8%)
13 (72.2%)
18
T4
0 (0%)
6 (100%)
6
N0
21 (50%)
21 (50%)
42
N1
3 (17.6%)
14 (82.4%)
17
N2
3 (20%)
12 (80%)
15
N3
0 (0%)
5 (100%)
5
Nx
6 (42.9%)
8 (57.1%)
14
Missing
0 (0%)
1 (100%)
1
M0
33 (35.5%)
60 (64.5%)
93
M1
0 (0%)
1 (100%)
1
Low (%)
High (%)
Male
18 (34%)
Female
0.73 0.023 0.003
0.001
0.014
0.46
Figure legends Fig. 1. Nrf2 is overexpressed in primary lung cancer samples and predicts poor prognosis. (A) The protein levels of Nrf2 in tumor and paired adjacent nontumor tissues in lung cancer patients. To investigate if Nrf2 is up-regulated in human lung cancer tissues, the protein levels of Nrf2 in tissues microarray which contains lung cancer tissues and paired adjacent lung tissues were detected by IHC staining. (B) The percentage of patients with down-regulated, up-regulated and no change of Nrf2 levels in tumor tissues compared with paired adjacent nontumor tissues. (C) Representative IHC images showing assigned intensity scores (0=Absence/faint; 1=Weak; 2=Moderate; 3=Strong) of Nrf2 staining. The intensity of Nrf2 staining was scored from 0 to 3 and grouped into low (score = 0, 1) and high (score = 2, 3) expression. (D) Correlation between histological type and Nrf2 protein expression. (E) Kaplan–Meier survival analysis of 94 patients based on Nrf2 expression in tumor tissues. (F) The protein levels of Nrf2 in A549, H460, H1299, BEAS-2B and 16HBE cells were detected by western blot. (G) The mRNA levels of Nrf2 target genes in A549, H460, H1299, BEAS-2B and 16HBE cells were detected by qRT-PCR. The colors of the heatmap reflect log2 (expression levels of Nrf2 target genes). All images are shown at ×400. Data are expressed as mean ± SD, n=3. ***P<0.001 represents significant difference.
Fig. 2. The expression of Nrf2 is associated with sensitivity of lung cancer cells to chemotherapeutic drugs. (A) Nrf2 shRNA was used to silence the expression of Nrf2 in A549 cells, and western blot was used to determine the protein levels of Nrf2. (B) Cytotoxic effects of chemotherapeutic drugs (cisplatin, doxorubicin and 5-FU) in A549 cells with or without Nrf2 knockdown. (C) Nrf2 shRNA was used to silence the expression of Nrf2 in H460 cells, and western blot was used to detect the protein levels of Nrf2. (D) Cytotoxic effects of chemotherapeutic drugs (cisplatin, doxorubicin and 5-FU) in H460 cells with or without Nrf2 knockdown. (E) Nrf2 overexpression plasmids were used to overexpress the expression of Nrf2 in A549 cells, and western blot was used to detect the protein levels of Nrf2. (F) Cytotoxic
effects of chemotherapeutic drugs (cisplatin, doxorubicin and 5-FU) in A549 cells with or without Nrf2 overexpression. (G) Nrf2 overexpression plasmids were used to overexpress the expression of Nrf2 in H460 cells, and western blot was used to detect the protein levels of Nrf2. (H) Cytotoxic effects of chemotherapeutic drugs (cisplatin, doxorubicin and 5-FU) in H460 cells with or without Nrf2 overexpression. Data are expressed as mean ± SD, n=3. ***P<0.001 vs. shControl group or vector group.
Fig. 3. Flumethasone inhibits Nrf2 protein levels in A549 and H460 cells. (A-B) A549 and H460 cells were treated with different concentrations of flumethasone (0, 50, 100, 200 nM) for 24 h, and western blot was used to detect the protein levels of Nrf2. (C-D) A549 and H460 cells were treated with 100 nM of flumethasone for 6 h, 12h and 24 h, and western blot was used to detect the protein levels of Nrf2. (E-F) Immunofluorescence staining. A549 and H460 cells were treated with 100 nM of flumethasone for 24 h, and protein levels of Nrf2 in cells were detected by immunofluorescence staining. All images are shown at ×200. Data are expressed as mean ± SD, n=3. ***P<0.001 vs. control group.
Fig. 4. Flumethasone reduces Nrf2 protein levels by promoting Nrf2 protein degradation in A549 and H460 cells. (A-D) A549 and H460 cells were treated with 100 nM of flumethasone, and qRT-PCR was used to detect mRNA levels of Nrf2 and Keap1 in cells. (E-F) A549 and H460 cells were treated with 100 nM of flumethasone, 20 µM of MG132, or combination of flumethasone and MG132 for 4 h, and the protein levels of Nrf2 were detected by western blot. (G-H) CHX-chase analysis. A549 and H460 cells were pre-treated with or without 100 nM of flumethasone for 4 h. Subsequently, cells were incubated with 25 µM of cycloheximide, and the protein levels of Nrf2 at indicated time points were detected by western blot. (I-J) Ubiquitination assay. A549 and H460 cells were treated with or without 100 nM of flumethasone for 4 h, and ubiquitination levels of Nrf2 in cells were detected by ubiquitination assay. Data are expressed as mean ± SD, n=3. ***P<0.001 vs. control group. N.S., no significant.
Fig. 5. Flumethasone sensitizes lung cancer cells with high Nrf2 expression to chemotherapeutic drugs. (A) The protein levels of Nrf2, NQO1 and HO-1 in A549, H460, H1299, BEAS-2B and 16HBE cells treated with or without 100 nM of flumethasone were detected by western blot. (B) The mRNA levels of Nrf2 target genes in A549, H460, H1299, BEAS-2B and 16HBE cells treated with or without 100 nM of flumethasone were detected by qRT-PCR. The colors of the heatmap reflect log2 (expression levels of Nrf2 target genes). (C) Effects of flumethasone on the sensitivity of A549, H460, H1299, BEAS-2B and 16HBE cells to cisplatin. (D) Effects of flumethasone on the sensitivity of A549, H460, H1299, BEAS-2B and 16HBE cells to doxorubicin. (E) Effects of flumethasone on the sensitivity of A549, H460, H1299, BEAS-2B and 16HBE cells to 5-FU. Data are expressed as mean ± SD, n=3. **P<0.01, ***P<0.001 vs. control group. N.S., no significant.
Fig. 6. Flumethasone enhances the sensitivity of A549 and H460 cells to chemotherapeutic drugs by inhibiting Nrf2 signaling. (A) A549 cells with or without Nrf2 knockdown were treated with 100 nM of flumethasone, and the protein levels of Nrf2, NQO1 and HO-1 were detected by western blot. (B) Effects of flumethasone on the sensitivity of A549 cells with or without Nrf2 knockdown to chemotherapeutic drugs (cisplatin, doxorubicin and 5-FU). (C) H460 cells with or without Nrf2 knockdown were treated with 100 nM of flumethasone, and the protein levels of Nrf2, NQO1 and HO-1 were detected by western blot. (D) Effects of flumethasone on the sensitivity of H460 cells with or without Nrf2 knockdown to chemotherapeutic drugs (cisplatin, doxorubicin and 5-FU). (E) Effects of Nrf2 overexpression plasmids on protein levels of Nrf2, NQO1 and HO-1 in flumethasone-treated A549 cells. (F) Effects of Nrf2 overexpression plasmids on flumethasone-induced chemosensitization in A549 cells. (G) Effects of Nrf2 overexpression plasmids on protein levels of Nrf2, NQO1 and HO-1 in flumethasone-treated H460 cells. (H) Effects of Nrf2 overexpression plasmids on flumethasone-induced chemosensitization in H460 cells. Data are expressed as mean ± SD, n=3. ***P<0.001 vs. control group. N.S., no
significant.
Fig. 7. Flumethasone enhances the sensitivity of A549 cells-derived xenografts to cisplatin by inhibiting Nrf2 signaling. (A) Sensitization of A549-shControl cells-derived xenografts to cisplatin treatment is induced by flumethasone. A549-shControl cells were injected into subdermal spaces of mice on the right flanks. When the volume of tumors reached 80-100 mm3, mice were randomly allocated into four groups, and then treated with vehicle, flumethasone (0.1 mg/kg), cisplatin (1 mg/kg) or in combination. Tumor volumes and body weights were recorded during the experiment. Tumors were excised, photographed and weighed at the end of the experiment. (B) No sensitization of A549-shNrf2 cells-derived xenografts to cisplatin treatment is induced by flumethasone. A549-shNrf2 cells were injected into subdermal spaces of mice on the right flanks. When the volume of tumors reached 80-100 mm3, mice were randomly allocated into four groups, and then treated with vehicle, flumethasone (0.1 mg/kg), cisplatin (1 mg/kg) or in combination. Tumor volumes and body weights were recorded during the experiment. Tumors were excised, photographed and weighed at the end of the experiment. (C-D) The protein levels of Nrf2, NQO1 and HO-1 in tumor tissues were detected by western blot. (E) IHC staining. The protein levels of Nrf2, NQO1, HO-1 and Ki67 in tumor tissues were detected by IHC staining. All images are shown at ×400. Data are expressed as mean ± SD, n=6. ***P<0.001 vs. vehicle group,
###
P<0.001 vs. cisplatin group. N.S.,
no significant.
Fig. 8. Toxicological assessment of flumethasone combined with cisplatin. (A) HE staining. The changes of pathological morphology of heart, liver, spleen, lung and kidney were evaluated by HE staining. (B) Organ indexes of heart, liver, spleen, lung and kidney. (C-E) ALT, AST and BUN levels in plasma from xenograft model mice in the vehicle, flumethasone, cisplatin and combination groups. All images are shown at ×400. Data are expressed as mean ± SD, n=12.
Fig. 9. Proposed model of regulation of Nrf2 signaling by flumethasone in lung cancer cells.
Highlights: 1. Flumethasone significantly inhibits Nrf2 signaling in A549 and H460 cells. 2. Flumethasone enhances sensitivity of A549 and H460 cells to chemotherapeutic drugs 3. The chemosensitization effect of flumethasone is mediated by Nrf2.
Conflicts of Interest Statement There is no conflict of interest.
S0304-3835(20)30018-5
DOI:
https://doi.org/10.1016/j.canlet.2020.01.010
Reference:
CAN 114652
To appear in:
Cancer Letters
Received Date: 23 September 2019 Revised Date:
9 January 2020
Accepted Date: 13 January 2020
Please cite this article as: Y. Zhou, Y. Zhou, K. Wang, T. Li, M. Yang, R. Wang, Y. Chen, M. Cao, R. Hu, Flumethasone enhances the efficacy of chemotherapeutic drugs in lung cancer by inhibiting Nrf2 signaling pathway, Cancer Letters, https://doi.org/10.1016/j.canlet.2020.01.010. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.
Abstract Nuclear factor erythroid-2-related factor 2 (Nrf2), a transcription factor, participates in protecting cells from electrophilic or oxidative stresses through regulating expression of cytoprotective and antioxidant genes. It has become one of the emerging targets for cancer chemosensitization, and small molecule inhibitors of Nrf2 can enhance the efficacy of chemotherapeutic drugs. Here, we found that flumethasone, a glucocorticoid, inhibited Nrf2 signaling in A549 and H460 cells by promoting Nrf2 protein degradation. Moreover, flumethasone significantly increased the sensitivity of A549 and H460 cells to chemotherapeutic drugs including cisplatin, doxorubicin and 5-FU. In mice bearing A549-shControl cells-derived xenografts, the size and weight of xenografts in the flumethasone and cisplatin combination group had a significant reduction compared with those in the cisplatin group, while in mice bearing A549-shNrf2 cells-derived xenografts, the size and weight of the xenografts in the combination group had no significant difference compared with those in the cisplatin group, demonstrating that chemosensitization effect of flumethasone is Nrf2-dependent. This work suggests that flumethasone can potentially be used as an adjuvant sensitizer to enhance the efficacy of chemotherapeutic drugs in lung cancer.
Keywords: Flumethasone, Nrf2, lung cancer, chemosensitization
Flumethasone enhances the efficacy of chemotherapeutic drugs in lung cancer by inhibiting Nrf2 signaling pathway Yunjiang Zhoua, Yang Zhoua, Keke Wanga, Tao Lia, Mengdi Yanga, Rui Wanga, Yaxin Chena, Mengran Caoa, Rong Hua, *
a
State Key Laboratory of Natural Medicines, School of Basic Medicine and
Clinical Pharmacy, China Pharmaceutical University, Nanjing, Jiangsu, China
* To whom correspondence should be addressed. School of Basic Medicine and Clinical Pharmacy, China Pharmaceutical University, 24 Tongjia Xiang, Nanjing, Jiangsu 210009, China. Email: [email protected] (Rong Hu).
Abstract Nuclear factor erythroid-2-related factor 2 (Nrf2), a transcription factor, participates in protecting cells from electrophilic or oxidative stresses through regulating expression of cytoprotective and antioxidant genes. It has become one of the emerging targets for cancer chemosensitization, and small molecule inhibitors of Nrf2 can enhance the efficacy of chemotherapeutic drugs. Here, we found that flumethasone, a glucocorticoid, inhibited Nrf2 signaling in A549 and H460 cells by promoting Nrf2 protein degradation. Moreover, flumethasone significantly increased the sensitivity of A549 and H460 cells to chemotherapeutic drugs including cisplatin, doxorubicin and 5-FU. In mice bearing A549-shControl cells-derived xenografts, the size and weight of xenografts in the flumethasone and cisplatin combination group had a significant reduction compared with those in the cisplatin group, while in mice bearing A549-shNrf2 cells-derived xenografts, the size and weight of the xenografts in the combination group had no significant difference compared with those in the cisplatin group, demonstrating that chemosensitization effect of flumethasone is Nrf2-dependent. This work suggests that flumethasone can potentially be used as an adjuvant sensitizer to enhance the efficacy of chemotherapeutic drugs in lung cancer.
Keywords: Flumethasone, Nrf2, lung cancer, chemosensitization
Abbreviations: Nrf2, nuclear factor erythroid 2-related factor 2; Keap1, Kelch-like ECH-associated protein 1; ARE, antioxidant response element; FLM, flumethasone; NQO1, NAD(P)H quinone oxidoreductase 1; HO-1, heme oxygenase-1; mRNA, messenger RNA; shRNA, short hairpin RNA; qRT-PCR, quantitative reverse transcription-PCR; IHC, immunohistochemistry; 8-OHdG, 8-hydroxydeoxyguanosine; GCR, glucocorticoid receptor; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen
1. Introduction Lung cancer is one of the most common malignant tumors and the leading cause of cancer-related death in the world [1]. Lung cancer is generally hard to be detected in the early stage, and therefore, patients are often diagnosed at mid or late stages. Although many chemotherapeutic drugs have been developed to treat lung cancer, the death rate is still rising. The main reason is that patients often develop chemoresistance during the course of chemotherapy. Therefore, it is urgent to explore the mechanism of chemoresistance and search for safe and effective chemotherapeutic sensitizers to increase the efficacy of chemotherapeutic drugs. Nrf2 participates in protecting cells from electrophilic or oxidative stresses by up-regulating expression of cytoprotective and antioxidant genes [2, 3]. Under normal conditions, Nrf2 is located in the cytoplasm and binds to the Kelch domain of Kelch-like ECH-associated protein 1 (Keap1) through the ETGE and DLG motifs of its Neh2 domain. Meanwhile, binding of Keap1 to Cul3-Rbx1-E3 ligase results in ubiquitination of Nrf2, which leads to proteasomal degradation of Nrf2 protein [4-6]. Therefore, Nrf2 protein is usually maintained at a low level in the cytoplasm. Under oxidative or electrophilic pressure, modification of Keap1 at cysteine residues blocks its interaction with Nrf2, leading to reducing Keap1-mediated degradation of Nrf2 [7]. Subsequently, Nrf2 accumulates in the cytoplasm and translocates into the nucleus, where it forms heterodimer with sMaf protein, which then binds to antioxidant response element (ARE) in the promoter region of Nrf2 target genes to promote their transcription [2, 8, 9]. Recent studies have shown that Nrf2 is overexpressed in a variety of cancers [10-13]. Meanwhile, high mutation rate of Keap1 is found in tumor tissues of lung cancer patients and such mutations lead to loss of Keap1 function and activate the Nrf2
signaling,
which
promote
cancer
progression,
chemoresistance
and
radioresistance [14-18]. Moreover, silencing Nrf2 or overexpressing Keap1 can increase the efficacy of chemotherapeutic drugs, and overexpression of Nrf2 will reduce it [19-22]. Therefore, Nrf2 may be a promising target for enhancing the
efficacy of chemotherapeutic drugs and combination of Nrf2 inhibitors with chemotherapeutic drugs may be an effective treatment for cancers. In this study, we identified flumethasone (FLM), a glucocorticoid with anti-inflammatory, vasoconstrictive and anti-hyperplasia properties, as a novel Nrf2 inhibitor. Furthermore, flumethasone could enhance the sensitivity of lung cancer to chemotherapeutic drugs. Therefore, flumethasone may be a promising agent to enhance the efficacy of chemotherapeutic drugs in lung cancer.
2. Materials and methods 2.1. Materials See supplementary materials for more details.
2.2. Cell culture and MTT assay All experimental procedures were performed using a standard protocol. See supplementary materials and methods for detailed experimental procedures.
2.3. Western blot assay, immunofluorescence assay, quantitative real-time PCR assay, CHX-chase analysis and ubiquitination assay All experimental procedures were performed using a standard protocol. See supplementary materials and methods for detailed experimental procedures.
2.4. Transfection of Nrf2 shRNA and Nrf2 plasmid All experimental procedures were performed using a standard protocol. See supplementary materials and methods for detailed experimental procedures.
2.5. Animal treatment Female BALB/c nude mice (18 ± 2 g, 6 weeks old) were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd (Beijing, China). Animal experiments were approved by the Animal Ethics Committee of China Pharmaceutical
University (Ethic approval number: 2019-11-003) and carried out following the guidelines in the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. See supplementary materials and methods for detailed animal experimental procedures.
2.6.
Immunohistochemistry
(IHC),
HE
staining
and
determination
of
8-hydroxydeoxyguanosine (8-OHdG) All experimental procedures were performed using a standard protocol. See supplementary materials and methods for detailed experimental procedures.
2.7. Human lung cancer tissue microarray A human lung cancer tissues microarray was purchased from Shanghai Outdo Biotech (Shanghai, China), which contains 94 cases of non-small-cell lung cancer tissues, with 86 cases having adjacent tissues. All patients had been pathologically diagnosed with non-small-cell lung cancer. IHC staining was used to analyze the expression of Nrf2 in tissues.
2.8. Statistical analysis The χ2 test was used to analyze the association between Nrf2 expression and clinicopathologic variables and Kaplan-Meier analysis was used to analyze the relationship between Nrf2 expression and patient survival. The other results were expressed as mean ± SD and were representative of three independent experiments. Statistical analysis was performed with the t-test for two groups or one-way ANOVA for multiple groups by using SPSS statistical software. P<0.05 was considered significant.
3. Results 3.1. Nrf2 is overexpressed in primary lung cancer samples and predicts poor prognosis
IHC staining was used to detect Nrf2 protein levels in lung cancer tissues microarray, and it was found that Nrf2 protein levels were higher in cancer tissues when compared with those in adjacent tissues (Fig. 1A). Moreover, about 67.4% of lung cancer patients had higher Nrf2 protein level in tumor tissues than in adjacent tissues (Fig. 1B). Among all lung cancer tissues in this tissue array, 64.9% had high Nrf2 expression (IHC scores 2 or 3, Fig. 1D), while in all adjacent tissues, 2.3% had high Nrf2 expression (IHC scores 2 or 3, Fig. 1D). In addition, the correlation between Nrf2 expression and patients’ survival was analyzed and it was found that lung cancer patients with high Nrf2 expression showed poor prognosis (Fig. 1E). Moreover, Nrf2 expression was correlated with the clinicopathological characteristics such as tumor size, clinical stage, T status and N status (Table 1). These results suggest that Nrf2 is overexpressed in primary lung cancer tissues and predicts poor prognosis. Next, the protein levels of Nrf2 in human lung cancer cells (A549, H460 and H1299) and normal bronchial epithelial cells (BEAS-2B and 16HBE) were detected by using western blot and it was found that Nrf2 protein levels in lung cancer cells were markedly higher than those in normal bronchial epithelial cells (Fig. 1F). Moreover, the mRNA levels of Nrf2 target genes in lung cancer cells were higher than those in normal bronchial epithelial cells (Fig. 1G). These data demonstrate that Nrf2 signaling is activated in lung cancer.
3.2. The expression of Nrf2 is associated with sensitivity of lung cancer cells to chemotherapeutic drugs To investigate whether Nrf2 expression is associated with the efficacy of chemotherapeutic drugs, Nrf2 shRNAs (shNrf2 #1 and shNrf2 #2) and Nrf2 overexpression plasmids were used to silence and overexpress Nrf2 in A549 and H460 cells respectively. We found that Nrf2 shRNAs decreased and Nrf2 overexpression plasmid increased the expression of Nrf2 in A549 and H460 cells (Fig. 2A, C, E, G and Supplementary Fig. S1). Moreover, the silencing efficiency of shNrf2 #2 was slightly stronger than that of shNrf2 #1 (Supplementary Fig. S1). Therefore, shNrf2 #2 was used to silence Nrf2 in the subsequent experiments. MTT assay was
then performed to detect the cytotoxicity of chemotherapeutic drugs including cisplatin, doxorubicin and 5-FU in A549 and H460 cells with Nrf2 knockdown or overexpression. The results showed that the sensitivity of A549 and H460 cells with Nrf2 knockdown to chemotherapeutic drugs was significantly increased (Fig. 2B, D), while the sensitivity of A549 and H460 with Nrf2 overexpression was significantly decreased (Fig. 2F, H), suggesting that Nrf2 expression is reversely correlated with the efficacy of chemotherapeutic drugs.
3.3. Flumethasone inhibits Nrf2 protein levels in A549 and H460 cells The inhibition on Nrf2 protein expression by flumethasone was then detected in A549 and H460 cells. As shown in Fig. 3A-B, 50, 100 and 200 nM of flumethasone significantly inhibited Nrf2 protein levels in cells. Moreover, it was found that Nrf2 protein levels in cells were significantly reduced at 6, 12 and 24 h after 100 nM of flumethasone treatment (Fig. 3C-D). In addition, endogenous Nrf2 immunostaining further confirmed that flumethasone treatment reduced the Nrf2 protein levels in A549 and H460 cells (Fig. 3E-F).
3.4. Flumethasone reduces Nrf2 protein levels by promoting Nrf2 protein degradation in A549 and H460 cells. The mRNA levels of Nrf2 and Keap1 in A549 and H460 cells after flumethasone treatment were detected by qRT-PCR, and the results showed that there was no significant difference in the mRNA levels of Nrf2 and Keap1 between flumethasone treated cells vs. control (Fig. 4A-D), suggesting that flumethasone does not inhibit Nrf2 at the transcription level. Therefore, we investigated whether flumethasone reduces Nrf2 protein level by promoting Nrf2 degradation. As shown in Fig. 4E-F, the treatment of MG132 (a proteasomal inhibitor) blocked the inhibitory effect of flumethasone on Nrf2 expression. Moreover, we found that flumethasone could significantly shorten the degradation half-life of Nrf2 (Fig. 4G-H), while ubiquitination analysis showed that ubiquitination levels of Nrf2 were significantly increased in cells after treatment with flumethasone (Fig. 4I-J). In the meantime,
ubiquitination levels of Keap1 in cells did no change significantly after treatment with flumethasone (Fig. 4I-J), indicating that flumethasone-induced degradation of Nrf2 protein is Keap1-independent. These results suggest that flumethasone reduces Nrf2 protein levels by promoting Nrf2 protein degradation in A549 and H460 cells. To investigate the role of glucocorticoid receptor (GCR) in flumethasone-induced down-regulation of Nrf2, two specific glucocorticoid receptor shRNAs (shGCR #1 and shGCR #2) were used to silence GCR expression in A549 and H460 cells. It was found that GCR knockdown blocked the inhibitory effects of flumethasone on Nrf2 (Supplementary Fig. S2A-B), indicating that flumethasone-induced down-regulation of Nrf2 is GCR-dependent. We also examined the effect of flumethasone on ubiquitination of Nrf2 in A549 and H460 cells with GCR knockdown and found that flumethasone could not significantly promote ubiquitination of Nrf2 in A549 and H460 cells with GCR knockdown (Supplementary Fig. S2C-D), indicating that flumethasone-mediated Nrf2 protein degradation is GCR-dependent.
3.5. Flumethasone sensitizes lung cancer cells with high Nrf2 expression to chemotherapeutic drugs Next, we investigated the inhibition on Nrf2 protein and its target genes expression by flumethasone treatment in A549, H460, H1299, BEAS-2B and 16HBE cells, and found that flumethasone significantly inhibited the protein levels of Nrf2 and its target genes in A549 and H460 cells, with a weaker inhibition in H1299 cells, and no effect in BEAS-2B and 16HBE cells (Fig. 5A-B). When flumethasone was used in combination with cisplatin, doxorubicin or 5-FU in A549, H460, H1299, BEAS-2B and 16HBE cells, it had strong chemosensitization effects in A549 and H460 cells, which highly express Nrf2, and relatively weak chemosensitization effects in H1299, BEAS-2B and 16HBE cells, with lower Nrf2 levels (Fig. 5C-E and Supplementary Table S2-4). These results demonstrate that flumethasone enhances sensitivity of lung cancer cells to chemotherapeutic drugs in those with relatively high Nrf2 levels.
3.6. Flumethasone enhances the sensitivity of A549 and H460 cells to chemotherapeutic drugs by inhibiting Nrf2 signaling To
investigate
whether
flumethasone-induced
chemosensitization
is
Nrf2-dependent, Nrf2 shRNA was used to silence Nrf2 expression in A549 and H460 cells, and then the cytotoxicity of chemotherapeutic drugs including cisplatin, doxorubicin and 5-FU in these cells were measured. When A549-shNrf2 and H460-shNrf2 cells were treated with 100 nM of flumethasone, Nrf2, NQO1 and HO-1 protein levels did not change significantly (Fig. 6A, C), and flumethasone could not increase the sensitivity of A549-shNrf2 and H460-shNrf2 cells to chemotherapeutic drugs (Fig. 6B, D). These data suggest that chemosensitization effect of flumethasone is dependent on its down-regulation of Nrf2 signaling. To further confirm this, Nrf2 overexpression plasmids were used to restore Nrf2 protein levels in cells (Fig. 6E, G), and it could eliminate flumethasone-induced chemosensitization in cells (Fig. 6F, H). These results further demonstrate that flumethasone-induced chemosensitization is Nrf2-dependent.
3.7. Flumethasone enhances the sensitivity of A549 cells-derived xenografts to cisplatin by inhibiting Nrf2 signaling To
investigate
whether
flumethasone-induced
chemosensitization
was
Nrf2-dependent in vivo, we constructed A549-shControl and A549-shNrf2 cells-derived xenografts mouse models. In A549-shControl cells-derived xenograft mice, the size and weight of xenografts in the flumethasone and cisplatin combination group had a significant reduction compared with those in the cisplatin group (Fig. 7A). However, in A549-shNrf2 cells-derived xenograft mice, the size and weight of the xenografts in the combination group had no significantly difference compared with those in the cisplatin group (Fig. 7B). Moreover, flumethasone could significantly inhibit Nrf2, NQO1 and HO-1 protein levels in A549-shControl cells-derived xenografts, while no inhibition was observed in A549-shNrf2 cells-derived xenografts (Fig. 7C-E). In addition, the levels of 8-OHdG and cleaved caspase-3 were also detected in tumor tissues and it was found that combination of flumethasone with
cisplatin significantly increased the levels of 8-OHdG and cleaved caspase-3 (Supplementary Fig. S3), indicating that flumethasone promotes cisplatin-induced apoptosis by increasing redox status. These results suggest that flumethasone enhances the sensitivity of A549 cells-derived xenografts to cisplatin by inhibiting Nrf2 signaling. We also investigated the toxicity of flumethasone and cisplatin alone or in combination in these mice. As shown in Fig. 7A-B, body weight of the mice did not change significantly during the experiment. Moreover, the pathological morphology and organ indexes of mice in the vehicle group, flumethasone group, cisplatin group and combination group had no significant difference (Fig. 8A-B). In addition, we examined the plasma levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and blood urea nitrogen (BUN) and found that there is no significant difference among all groups (Fig. 8C-E). Therefore, toxicity was not observed in mice at the concentrations used here in our study.
4. Discussion Chemoresistance is the major obstacle to successful treatment of cancers. Recent studies have demonstrated that overactivation of Nrf2 can lead to chemoresistance in lung cancer as well as other types of cancers [21, 23, 24]. By using Nrf2 siRNA to inhibit Nrf2 expression in cancer cells, researchers found that the growth of cancer cells was slowed and the sensitivity of cancer cells to chemotherapeutic drugs was increased [21]. Consistent with previous studies, we found that Nrf2 was highly expressed in tumor tissues of lung cancer patients and silencing Nrf2 in lung cancer cells could increase the sensitivity of these cells to chemotherapeutic drugs. Moreover, we also found that the expression of Nrf2 in tumor tissues of lung cancer patients was associated with prognosis and clinicopathological characteristics. These results indicate that Nrf2 may be an effective target in the treatment of lung cancer, and therefore, combination of Nrf2 inhibitors with chemotherapeutic drugs may be an useful strategy for the treatment of lung cancer.
In previous studies, small-molecule Nrf2 inhibitors, such as epigallocatechin 3-gallate [25], luteolin [26], alkaloid trigonelline [27], brusatol [28], halofuginone [29] and camptothecin [30], have been shown to increase chemosensitivity of cancers to chemotherapeutic drugs, indicating that they can potentially be used as chemotherapeutic sensitizers. We have previously reported that wogonin reverses doxorubicin resistance of MCF-7/DOX cells through suppressing Nrf2 signaling [31]. However, its clinical side effects are unclear. We also found that digoxin can reverse gemcitabine resistance in pancreatic cancer cells through inhibiting Nrf2 signaling [32], while long-term use of digoxin might cause cardiotoxicity in humans. In the present study, flumethasone was found to inhibit the activity of Nrf2 at nanomolar concentrations, and it sensitized lung cancer cells with high Nrf2 expression to chemotherapeutic drugs. Moreover, flumethasone-mediated chemosensitization of lung cancer relied on its ability to inhibit the Nrf2 signaling pathway. In our in vivo experiments, flumethasone and cisplatin in combination did not significantly change the body weight, organ indexes and pathological morphology in mice, indicating that combination of flumethasone and cisplatin at the doses used here does not have obvious toxicity. Therefore, flumethasone may be used as an adjuvant in lung cancer treatment. Both the transcription process of genes and the degradation process of proteins will affect the protein level in cells. In this study, we found that flumethasone treatment did not change Nrf2 mRNA levels in A549 and H460 cells, indicating that flumethasone does not regulate Nrf2 at transcription level. We then investigated whether flumethasone regulates Nrf2 protein degradation and found that flumethasone could decrease Nrf2 protein levels by promoting its degradation. It should be noted that Keap1 mutations were found in A549 and H460 cells, which led to the lack of interaction of Keap1-Nrf2 in these cells [14]. Therefore, Keap1-dependent Nrf2 protein degradation should be minimal in these cells, and we speculated that flumethasone-mediated
Nrf2
degradation
in
A549
and
H460
cells
are
Keap1-independent. Indeed, we found that flumethasone did not increase ubiquitination
of
Keap1
in
A549
and
H460
cells,
suggesting
that
flumethasone-mediated Nrf2 degradation is Keap1-independent. Previously reports showed that glucocorticoids inhibited Nrf2 signaling through the activation of GCR and potentiated the antitumor effects of chemotherapeutic drugs [33-35]. Here, we found
that
flumethasone-induced
down-regulation
of
Nrf2
protein
was
GCR-dependent. It has been reported that β-TrCP and Hrd1 are involved in regulating the degradation of Nrf2 in a Keap1-independent manner under certain circumstances [36-38]. However, it is not clear whether β-TrCP and Hrd1 are also involved in the flumethasone-mediated regulation of Nrf2 degradation in A549 and H460 cells. In summary, this study demonstrates that flumethasone inhibits Nrf2 signaling pathway through promoting degradation of Nrf2 protein and enhances the efficacy of chemotherapeutic drugs in lung cancer cells (Fig. 9). We also provided in vitro and in vivo evidences that flumethasone in combination with cisplatin could be a potential therapeutic strategy in treating lung cancer which highly express Nrf2.
Acknowledgements This work was supported by the National Natural Science Foundation of China (No.81372268, No.81672816 and No.81872337), the Program for Jiangsu Province Innovative Research (KYLX16_1120), the Natural Science Foundation for Distinguished Young Scholars of Jiangsu Province (No. BK20130026).
Conflict of interest There is no conflict of interest.
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Table 1 Association between clinicopathologic variables of lung cancer patients and Nrf2 expression. Variables
Group
Sex Age Tumor size Stage
T status
N status
M status
Nrf2 expression
Total
P-value
35 (66%)
53
0.792
15 (36.6%)
26 (63.4%)
41
≤ 60 years
18 (36.7%)
31 (63.3%)
49
> 60 years
15 (33.3%)
30 (66.7%)
45
≤ 3 cm
17 (50%)
17 (50%)
34
> 3 cm
16 (26.7%)
44 (73.3%)
60
Ⅰ
17 (56.7%)
13 (43.3%)
30
Ⅰ-Ⅰ
16 (25.4%)
47 (74.6%)
63
Missing
0 (0%)
1 (100%)
1
T1
14 (70%)
6 (30%)
20
T2
14 (28%)
36 (72%)
50
T3
5 (27.8%)
13 (72.2%)
18
T4
0 (0%)
6 (100%)
6
N0
21 (50%)
21 (50%)
42
N1
3 (17.6%)
14 (82.4%)
17
N2
3 (20%)
12 (80%)
15
N3
0 (0%)
5 (100%)
5
Nx
6 (42.9%)
8 (57.1%)
14
Missing
0 (0%)
1 (100%)
1
M0
33 (35.5%)
60 (64.5%)
93
M1
0 (0%)
1 (100%)
1
Low (%)
High (%)
Male
18 (34%)
Female
0.73 0.023 0.003
0.001
0.014
0.46
Figure legends Fig. 1. Nrf2 is overexpressed in primary lung cancer samples and predicts poor prognosis. (A) The protein levels of Nrf2 in tumor and paired adjacent nontumor tissues in lung cancer patients. To investigate if Nrf2 is up-regulated in human lung cancer tissues, the protein levels of Nrf2 in tissues microarray which contains lung cancer tissues and paired adjacent lung tissues were detected by IHC staining. (B) The percentage of patients with down-regulated, up-regulated and no change of Nrf2 levels in tumor tissues compared with paired adjacent nontumor tissues. (C) Representative IHC images showing assigned intensity scores (0=Absence/faint; 1=Weak; 2=Moderate; 3=Strong) of Nrf2 staining. The intensity of Nrf2 staining was scored from 0 to 3 and grouped into low (score = 0, 1) and high (score = 2, 3) expression. (D) Correlation between histological type and Nrf2 protein expression. (E) Kaplan–Meier survival analysis of 94 patients based on Nrf2 expression in tumor tissues. (F) The protein levels of Nrf2 in A549, H460, H1299, BEAS-2B and 16HBE cells were detected by western blot. (G) The mRNA levels of Nrf2 target genes in A549, H460, H1299, BEAS-2B and 16HBE cells were detected by qRT-PCR. The colors of the heatmap reflect log2 (expression levels of Nrf2 target genes). All images are shown at ×400. Data are expressed as mean ± SD, n=3. ***P<0.001 represents significant difference.
Fig. 2. The expression of Nrf2 is associated with sensitivity of lung cancer cells to chemotherapeutic drugs. (A) Nrf2 shRNA was used to silence the expression of Nrf2 in A549 cells, and western blot was used to determine the protein levels of Nrf2. (B) Cytotoxic effects of chemotherapeutic drugs (cisplatin, doxorubicin and 5-FU) in A549 cells with or without Nrf2 knockdown. (C) Nrf2 shRNA was used to silence the expression of Nrf2 in H460 cells, and western blot was used to detect the protein levels of Nrf2. (D) Cytotoxic effects of chemotherapeutic drugs (cisplatin, doxorubicin and 5-FU) in H460 cells with or without Nrf2 knockdown. (E) Nrf2 overexpression plasmids were used to overexpress the expression of Nrf2 in A549 cells, and western blot was used to detect the protein levels of Nrf2. (F) Cytotoxic
effects of chemotherapeutic drugs (cisplatin, doxorubicin and 5-FU) in A549 cells with or without Nrf2 overexpression. (G) Nrf2 overexpression plasmids were used to overexpress the expression of Nrf2 in H460 cells, and western blot was used to detect the protein levels of Nrf2. (H) Cytotoxic effects of chemotherapeutic drugs (cisplatin, doxorubicin and 5-FU) in H460 cells with or without Nrf2 overexpression. Data are expressed as mean ± SD, n=3. ***P<0.001 vs. shControl group or vector group.
Fig. 3. Flumethasone inhibits Nrf2 protein levels in A549 and H460 cells. (A-B) A549 and H460 cells were treated with different concentrations of flumethasone (0, 50, 100, 200 nM) for 24 h, and western blot was used to detect the protein levels of Nrf2. (C-D) A549 and H460 cells were treated with 100 nM of flumethasone for 6 h, 12h and 24 h, and western blot was used to detect the protein levels of Nrf2. (E-F) Immunofluorescence staining. A549 and H460 cells were treated with 100 nM of flumethasone for 24 h, and protein levels of Nrf2 in cells were detected by immunofluorescence staining. All images are shown at ×200. Data are expressed as mean ± SD, n=3. ***P<0.001 vs. control group.
Fig. 4. Flumethasone reduces Nrf2 protein levels by promoting Nrf2 protein degradation in A549 and H460 cells. (A-D) A549 and H460 cells were treated with 100 nM of flumethasone, and qRT-PCR was used to detect mRNA levels of Nrf2 and Keap1 in cells. (E-F) A549 and H460 cells were treated with 100 nM of flumethasone, 20 µM of MG132, or combination of flumethasone and MG132 for 4 h, and the protein levels of Nrf2 were detected by western blot. (G-H) CHX-chase analysis. A549 and H460 cells were pre-treated with or without 100 nM of flumethasone for 4 h. Subsequently, cells were incubated with 25 µM of cycloheximide, and the protein levels of Nrf2 at indicated time points were detected by western blot. (I-J) Ubiquitination assay. A549 and H460 cells were treated with or without 100 nM of flumethasone for 4 h, and ubiquitination levels of Nrf2 in cells were detected by ubiquitination assay. Data are expressed as mean ± SD, n=3. ***P<0.001 vs. control group. N.S., no significant.
Fig. 5. Flumethasone sensitizes lung cancer cells with high Nrf2 expression to chemotherapeutic drugs. (A) The protein levels of Nrf2, NQO1 and HO-1 in A549, H460, H1299, BEAS-2B and 16HBE cells treated with or without 100 nM of flumethasone were detected by western blot. (B) The mRNA levels of Nrf2 target genes in A549, H460, H1299, BEAS-2B and 16HBE cells treated with or without 100 nM of flumethasone were detected by qRT-PCR. The colors of the heatmap reflect log2 (expression levels of Nrf2 target genes). (C) Effects of flumethasone on the sensitivity of A549, H460, H1299, BEAS-2B and 16HBE cells to cisplatin. (D) Effects of flumethasone on the sensitivity of A549, H460, H1299, BEAS-2B and 16HBE cells to doxorubicin. (E) Effects of flumethasone on the sensitivity of A549, H460, H1299, BEAS-2B and 16HBE cells to 5-FU. Data are expressed as mean ± SD, n=3. **P<0.01, ***P<0.001 vs. control group. N.S., no significant.
Fig. 6. Flumethasone enhances the sensitivity of A549 and H460 cells to chemotherapeutic drugs by inhibiting Nrf2 signaling. (A) A549 cells with or without Nrf2 knockdown were treated with 100 nM of flumethasone, and the protein levels of Nrf2, NQO1 and HO-1 were detected by western blot. (B) Effects of flumethasone on the sensitivity of A549 cells with or without Nrf2 knockdown to chemotherapeutic drugs (cisplatin, doxorubicin and 5-FU). (C) H460 cells with or without Nrf2 knockdown were treated with 100 nM of flumethasone, and the protein levels of Nrf2, NQO1 and HO-1 were detected by western blot. (D) Effects of flumethasone on the sensitivity of H460 cells with or without Nrf2 knockdown to chemotherapeutic drugs (cisplatin, doxorubicin and 5-FU). (E) Effects of Nrf2 overexpression plasmids on protein levels of Nrf2, NQO1 and HO-1 in flumethasone-treated A549 cells. (F) Effects of Nrf2 overexpression plasmids on flumethasone-induced chemosensitization in A549 cells. (G) Effects of Nrf2 overexpression plasmids on protein levels of Nrf2, NQO1 and HO-1 in flumethasone-treated H460 cells. (H) Effects of Nrf2 overexpression plasmids on flumethasone-induced chemosensitization in H460 cells. Data are expressed as mean ± SD, n=3. ***P<0.001 vs. control group. N.S., no
significant.
Fig. 7. Flumethasone enhances the sensitivity of A549 cells-derived xenografts to cisplatin by inhibiting Nrf2 signaling. (A) Sensitization of A549-shControl cells-derived xenografts to cisplatin treatment is induced by flumethasone. A549-shControl cells were injected into subdermal spaces of mice on the right flanks. When the volume of tumors reached 80-100 mm3, mice were randomly allocated into four groups, and then treated with vehicle, flumethasone (0.1 mg/kg), cisplatin (1 mg/kg) or in combination. Tumor volumes and body weights were recorded during the experiment. Tumors were excised, photographed and weighed at the end of the experiment. (B) No sensitization of A549-shNrf2 cells-derived xenografts to cisplatin treatment is induced by flumethasone. A549-shNrf2 cells were injected into subdermal spaces of mice on the right flanks. When the volume of tumors reached 80-100 mm3, mice were randomly allocated into four groups, and then treated with vehicle, flumethasone (0.1 mg/kg), cisplatin (1 mg/kg) or in combination. Tumor volumes and body weights were recorded during the experiment. Tumors were excised, photographed and weighed at the end of the experiment. (C-D) The protein levels of Nrf2, NQO1 and HO-1 in tumor tissues were detected by western blot. (E) IHC staining. The protein levels of Nrf2, NQO1, HO-1 and Ki67 in tumor tissues were detected by IHC staining. All images are shown at ×400. Data are expressed as mean ± SD, n=6. ***P<0.001 vs. vehicle group,
###
P<0.001 vs. cisplatin group. N.S.,
no significant.
Fig. 8. Toxicological assessment of flumethasone combined with cisplatin. (A) HE staining. The changes of pathological morphology of heart, liver, spleen, lung and kidney were evaluated by HE staining. (B) Organ indexes of heart, liver, spleen, lung and kidney. (C-E) ALT, AST and BUN levels in plasma from xenograft model mice in the vehicle, flumethasone, cisplatin and combination groups. All images are shown at ×400. Data are expressed as mean ± SD, n=12.
Fig. 9. Proposed model of regulation of Nrf2 signaling by flumethasone in lung cancer cells.
Highlights: 1. Flumethasone significantly inhibits Nrf2 signaling in A549 and H460 cells. 2. Flumethasone enhances sensitivity of A549 and H460 cells to chemotherapeutic drugs 3. The chemosensitization effect of flumethasone is mediated by Nrf2.
Conflicts of Interest Statement There is no conflict of interest.