β-cryptoxanthin restores nicotine-reduced lung SIRT1 to normal levels and inhibits nicotine-promoted lung tumorigenesis and emphysema in A/J mice

Abstract

Nicotine, a large constituent of cigarette smoke, is associated with an increased risk of lung cancer, but the data supporting this relationship are inconsistent. Here, we found that nicotine treatment not only induced emphysema but also increased both lung tumor multiplicity and volume in 4-nitrosamino-1-(3-pyridyl)-1-butanone (NNK)-initiated lung cancer in A/J mice. This tumor-promoting effect of nicotine was accompanied by significant reductions in survival probability and lung Sirtuin 1 (SIRT1) expression, which has been proposed as a tumor suppressor. The decreased level of SIRT1 was associated with increased levels of AKT phosphorylation and interleukin (il)-6 mRNA but decreased tumor suppressor p53 and retinoic acid receptor (RAR)-β mRNA levels in the lungs. Using this mouse model, we then determined whether β-cryptoxanthin (BCX), a xanthophyll that is strongly associated with a reduced risk of lung cancer in several cohort studies, can inhibit nicotine-induced emphysema and lung tumorigenesis. We found that BCX supplementation at two different doses was associated with reductions of the nicotine-promoted lung tumor multiplicity and volume, as well as emphysema in mice treated with both NNK and nicotine. Moreover, BCX supplementation restored the nicotine-suppressed expression of lung SIRT1, p53, and RAR-β to that of the control group, increased survival probability, and decreased the levels of lung il-6 mRNA and phosphorylation of AKT. The present study indicates that BCX is a preventive agent against emphysema and lung cancer with SIRT1 as a potential target. In addition, our study establishes a relevant animal lung cancer model for studying tumor growth within emphysematous microenvironments.

Figure 1. Study design and experiment schemes

The study design (top) and experiment scheme (bottom) of Experiment 1, which tested the effects of nicotine on promoting lung tumor and emphysema development (A), and Experiment 2, which tested the effect of BCX supplementation on the promotion of lung tumor and emphysema development (B) are shown. Abbreviations: BCX (10): β-cryptoxanthin at 10 mg/kg diet; BCX (20): β-cryptoxanthin at 20 mg/kg diet; C: control; i.p.: intraperitoneal; Nic: nicotine; NNK: 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; wk: week(s).

Figure 2. Nicotine decreases survival probability, lung tumor multiplicity and size, and emphysema development

The survival curves for the three treatment groups in Experiment 1 were analyzed using the SAS® PROC LIFETEST. The results of the log-rank test for the control, NNK, and NNK + Nic groups (p < 0.01) are shown in (A). The quantification of the lung tumor multiplicity is shown in (B). Inset: lung surface tumors (arrows). The number of lung surface tumors and the quantification of the lung tumor volume (mm³) are shown in (C). Inset: the measurement of the lung tumor diameter with a caliper. Representative images of the hematoxylin and eosin (H&E)-stained slides (grid: 0.5 × 0.5 μ-pixels) are shown in (D).The degree of emphysema was quantified by calculating the mean linear intercept (Lm) using 10 fields per animal (E). The lung il-6 mRNA levels are shown in (F). The lung cyclin D1 protein levels are shown in (G). Inset: a representative image of the cyclin D1 and GAPDH proteins. The lung p50 protein levels are shown in (H). Inset: a representative image of the p50 and GAPDH proteins. The lung egr1 mRNA levels are shown in (I). The proteins were detected using Western blotting. The intensity of the bands was quantified densitometry and was normalized to GAPDH. The mRNA levels were measured by qRT-PCR and normalized to β-actin mRNA levels. The columns represent the mean ± sem. *p < 0.05. Abbreviations: C: control; Nic: nicotine; NNK: 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; il-6: interleukin-6; egr1: early growth response 1.

Figure 3. Nicotine decreases the level of SIRT1 protein and the transcription of tumor suppressor genes, and increases AKT phosphorylation

The lung SIRT1 protein levels are shown in (A). Inset: a representative image of lung SIRT1 and GAPDH proteins. The lung sirt1 mRNA levels are shown in (B). The quantification of AKT phosphorylation on Ser473 is shown in (C). Inset: a representative image of phosphorylated AKT (pAKT) and total AKT. The mRNA levels of the p53 and RAR-β are shown in (D) and (E), respectively. The proteins were detected using Western blotting. The intensity of the bands was quantified using densitometry and normalized to GAPDH or total AKT. The mRNA levels were measured with qRT-PCR and normalized to β-actin mRNA levels. The columns represent the mean ± sem. *p < 0.05. Abbreviations: C: control; Nic: nicotine; NNK: 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; RAR-β: retinoic acid receptor-β, SIRT1: sirtuin 1.

Figure 4. BCX supplementation maintains survival probability, decreases lung tumor multiplicity and size, and prevents emphysema

The survival curves for the three treatment groups in Experiment 2 were analyzed using SAS® PROC LIFETEST. The log-rank test results for the control, NNK + Nic, NNK + Nic + BCX (10), and NNK + Nic + BCX (20) groups (P < 0.01) are shown in (A). The quantification of the lung tumor multiplicity is shown in (B). The number of lung suface tumors and the quantification of the lung tumor volume (mm³) are shown in (C). Effects of BCX supplementation on nicotine-induced emphysema and lung inflammation. The degree of emphysema was quantified by calculating the mean linear intercept (Lm) using 10 fields per animal (D). The lung il-6 mRNA levels are shown in (E). The lung cyclin D1 protein levels are shown in (F). Inset: a representative image of the cyclin D1 and GAPDH proteins. The lung p50 protein levels are shown in (G). Inset: a representative image of the p50 and GAPDH proteins. The lung egr1 mRNA levels are shown in (H). The proteins were detected using Western blotting. The intensity of the bands was quantified using a densitometry and normalized to GAPDH levels. The mRNA levels were measured using qRT-PCR and normalized to β-actin mRNA levels. The columns represent the mean ± sem. *p < 0.05. Abbreviations: BCX (10): β-cryptoxanthin at 10 mg/kg diet; BCX (20): β-cryptoxanthin at 20 mg/kg diet; C: control; Nic: nicotine; NNK: 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; il-6: interleukin-6; egr1: early growth response 1.

Figure 5. BCX restores the SIRT1 protein level and the transcription of tumor suppressor genes and decreases the phosphorylation of AKT

The lung SIRT1 protein levels are shown in (A). Inset: a representative image of the SIRT1 and GAPDH proteins. The lung sirt1 mRNA levels are shown in (B). The quantification of AKT phosphorylation on Ser473 is shown in (C). Inset: a representative image of phosphorylated AKT (pAKT) and total AKT. The mRNA levels of p53 and RAR-β are shown in (D) and (E), respectively. The proteins were detected using Western blotting. The intensity of the bands was quantified using densitometry and normalized to GAPDH or total AKT levels. The mRNA levels were measured using qRT-PCR and were normalized to β-actin mRNA levels. The columns represent the mean ± sem. *p < 0.05. Abbreviations: BCX (10): β-cryptoxanthin at 10 mg/kg diet; BCX (20): β-cryptoxanthin at 20 mg/kg diet; C: control; Nic: nicotine; NNK: 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; RAR-β: retinoic acid receptor-β, SIRT1: sirtuin 1.