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. 2012 Aug;6(4):405-17.
doi: 10.1016/j.molonc.2012.03.005. Epub 2012 Apr 2.

Curcumin inhibits tumor proliferation induced by neutrophil elastase through the upregulation of α1-antitrypsin in lung cancer

Yan Xu  1 , Jingjie Zhang, Jing Han, Xueyang Pan, Yajun Cao, Hao Guo, Yan Pan, Yu An, Xuejun Li
Affiliations

Curcumin inhibits tumor proliferation induced by neutrophil elastase through the upregulation of α1-antitrypsin in lung cancer

Yan Xu et al. Mol Oncol. 2012 Aug.

Abstract

Lung carcinogenesis is a complex process in an unregulated inflammatory environment. Curcumin has been extensively investigated as a multi-target anti-tumor and anti-inflammation compound. In this paper, we demonstrate a novel inflammation-related mechanism for curcumin-induced inhibition of lung tumor growth. We found that neutrophil elastase, an important regulator of inflammatory processes, directly triggered tumor cell proliferation in human lung adenocarcinoma A549 cells, and curcumin could completely suppress the excess tumor proliferation induced by neutrophil elastase. α1-antitrypsin is synthesized by tumor cells and is the natural inhibitor of neutrophil elastase. We found that curcumin counteracted the decrease of α1-antitrypsin induced by neutrophil elastase by inducing the promoter activity of α1-antitrypsin and promoting its expression in A549 cells. The inhibition of neutrophil elastase-induced proliferation by curcumin was dependent on the PI3K/Akt pathway. Knockdown of α1-antitrypsin by siRNA further enhanced the tumor cell proliferation induced by neutrophil elastase and significantly blocked the anti-proliferation effect of curcumin against neutrophil elastase. Curcumin remarkably inhibited the primary tumor growth of Lewis lung carcinoma (LLC) in C57BL/6 mice. We further showed that curcumin upregulated the level of α1-antitrypsin in primary tumor tissue by promoting its local expression, and the protein level of neutrophil elastase in tumor tissue was obviously decreased in mice treated with curcumin. Overall, our results suggest that neutrophil elastase and α1-antitrypsin play important roles in modulating lung tumor proliferation in inflammatory microenvironment and curcumin inhibits neutrophil elastase-induced tumor proliferation via upregulating α1-antitrypsin expression in vitro and in vivo.

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Figures

Figure 1
Figure 1
Curcumin prevented tumor cell proliferation induced by neutrophil elastase in A549 cells. (A) Cell viability detected by CCK‐8 assay for A549 cells stimulated with neutrophil elastase (NE). Data represent mean ± SEM, n = 3. *P < 0.05, **P < 0.01 vs. neutrophil elastase free group (control). (B) The effects of curcumin (Cur) on cell viability of A549 cells treated with or without neutrophil elastase. Data represent mean ± SEM, n = 3. *P < 0.05, ***P < 0.001 compared to the group without curcumin and neutrophil elastase. ###, P < 0.001 compared to the group with neutrophil elastase and without curcumin. (C) The effects of curcumin on the cell viability of Calu‐3 cells (K‐ras WT) induced by neutrophil elastase. Data represent mean ± SEM, n = 3. *P < 0.05, **P < 0.01. (D) Representative HCS images for BrdU, Ki‐67 and DAPI from A549 cells exposed to neutrophil elastase and curcumin. (E–H) Cell number (E), the ratio of the cells with 2N DNA content and those with 4N DNA content (F) and the quantification for BrdU (G) and Ki‐67 (H) were analyzed by HCS system. Data represent mean ± SEM, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 2
Figure 2
Curcumin‐induced α1‐antitrypsin expression in A549 cells provoked by neutrophil elastase. (A) Dose effects of curcumin (Cur) on α1‐antitrypsin (α1‐AT) protein levels in A549 cells when treated with or without neutrophil elastase (NE). (B) Western bolt of α1‐antitrypsin for 20 μM of curcumin‐treated and 40 nM of neutrophil elastase‐exposed A549 lysates. Data represent mean ± SEM, n = 3. **P < 0.01. (C) Confocal images for α1‐antitrypsin from A549 cells exposed to curcumin and neutrophil elastase. A549 cells were grown to 80% confluency and then incubated with 20 μM of curcumin and/or 40 nM of neutrophil elastase for 24 h. The cells were labeled by anti‐human α1‐antitrypsin antibody and DyLight 594‐conjugated secondary antibody (red) and Hoechst 33342 staining (blue). (D) Real‐time PCR for α1‐antitrypsin in A549 cells treated with curcumin and neutrophil elastase. Data represent mean ± SEM, n = 3. *P < 0.05, **P < 0.01. (E) The effect of curcumin on the normalized luciferase activity in A549 cells transfected with α1‐antitrypsin promoter when treated with neutrophil elastase. Data represent mean ± SEM, n = 3. *P < 0.05, **P < 0.01.
Figure 3
Figure 3
Curcumin attenuated neutrophil elastase‐induced proliferation depending on the PI3K/Akt pathway. (A) CCK‐8 assay for A549 cells stimulated with 40 nM of neutrophil elastase in the presence of 20 μM curcumin (Cur) and 10 μM LY294002 or 10 μM U0126, mean ± SEM, n = 3. *P < 0.05, ***P < 0.001. (B) Western blot for IRS‐1, pAkt, Akt, p‐mTOR, mTOR, pERK, ERK, p‐IKKβ, IKKβ, p‐p65, p65, Bcl‐2 and GAPDH in A549 cells treated with neutrophil elastase (NE) and curcumin. (C–I) Statistical data shown as the histograms of Western blot for IRS‐1 (C), the pAkt:Akt ratio (D), the p‐mTOR:mTOR ratio (E), the pERK:ERK ratio (F), the p‐IKKβ:IKKβ ratio (G), the p‐p65:p65 ratio (H) and Bcl‐2 (I) in A549 cells treated with neutrophil elastase and curcumin. Data represent mean ± SEM, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 4
Figure 4
α1‐antitrypsin knockdown partially inhibited curcumin‐induced suppression of cell proliferation in A549 cells exposed to neutrophil elastase. (A) Western blot to detect the efficiency of α1‐antitrypsin (α1‐AT) knockdown. (B) Cell viability in A549 cells transfected with or without siRNA against α1‐antitrypsin. Scramble (Scr) siRNA was used as control. A549 cells were transfected with or without siRNA against α1‐antitrypsin for 24 h, and then incubated with or without 20 μM of curcumin (Cur) and 40 nM of neutrophil elastase (NE) for additional 24 h. Data represent mean ± SEM, n = 3. *P < 0.05, ***P < 0.001. (C) Western blot for pAkt and Akt in α1‐antitrypsin‐scilenced A549 cells exposed to neutrophil elastase and curcumin. Data represent mean ± SEM, n = 3. *P < 0.05, ***P < 0.001.
Figure 5
Figure 5
The effect of curcumin on the primary tumor growth and lung metastasis of Lewis lung carcinoma (LLC). (A) Primary tumor weights of mice treated with the vehicle (control), 100 mg/kg curcumin and 300 mg/kg curcumin for 21 days after subcutaneous injection of LLC cells. n = 8–10 mice per group, mean ± SEM, **P < 0.01 vs. control. (B) Lung net weights for each group. n = 8–10 mice per group, mean ± SEM, **P < 0.01 vs. control. (C) Gross LLC lung metastasis for the three groups. n = 8–10 mice per group, mean ± SEM, **P < 0.01, ***P < 0.001 vs. control. (D) Representative Ki‐67 immunohistochemistry images and statistical analysis of Ki‐67 expression in primary tumor tissue for each group. Data are expressed as the relative percentage of the value of Ki‐67 content for the untreated control group. n = 6, mean ± SEM, **P < 0.01, ***P < 0.001 vs. control.
Figure 6
Figure 6
Curcumin‐induced increase of α1‐antitrypsin and decrease of neutrophil elastase in primary tumor tissue of C57BL/6 mice. (A) Western blot of α1‐antitrypsin (α1‐AT) in Lewis lung carcinoma tissue from the three groups. Data represent mean ± SEM, n = 6. **P < 0.01 as compared with control. (B) Quantitative real‐time PCR analysis for α1‐antitrypsin in tumor tissue. The mRNA level of α1‐antitrypsin was normalized to β‐actin. Data represent mean ± SEM, n = 3. **P < 0.01 as compared with control. (C) Western analysis of neutrophil elastase (NE) in tumor tissues. Data represent mean ± SEM, n = 6. *P < 0.05, ***P < 0.001 as compared with control. (D) Western blot for IRS‐1, pAkt, Akt, p‐mTOR, mTOR, pERK, ERK, p‐IKKβ, IKKβ, p‐p65, p65, Bcl‐2 and β‐actin in primary tumor tissues. (E) Statistical data shown as the histograms of Western blot for IRS‐1, the ratio of pAkt:Akt, p‐mTOR:mTOR and pERK:ERK. (F) Statistical data of Western blot for the p‐IKKβ:IKKβ ratio, the p‐p65:p65 ratio and Bcl‐2. Data represent mean ± SEM, n = 6. **P < 0.01, ***P < 0.001 as compared with control.
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