Estrogen Receptor Alpha Promoting NNK-induced Lung Cancer Carcinogenesis Depends On CYP1B1 And ERK

Object (1)To clarify the expression of ERa in lung tissues of lung cancer patients and A/J mice. (2) To prove both ERa and CYP1B1 are involved in NNK-induced lung cancer carcinogenesis. (3) To explore the underlying mechanism of ERa promoting NNK-induced lung cancer.Method Firstly, we examined the expression of ERa in human lung tumor and non-tumor tissues obtained from lung cancer patients by immunohistochemical staining(IHC). Secondly, we conducted animal work to detect the level of ERa in lung tissues of A/J mice by IHC, Western blot and real time-PCR. Brief steps as followed: 160 A/J mice, 6 weeks, were separated into two groups, experience group and control group. At the begining,mice of experience group were treated with NNK(100 mg/kg, i. p.), and the mice of control group were treated with PBS as same volume as NNK. Maintained for 26 weeks, 10 mice of each group were sacrificed by cervical vertebra dislocation, 4 week once time until terminal of experiment. The left side of lung tissues were fixed by formaldehyde solution and incised into 5 ?m sections for IHC and HE staining. The right side of lung tissues were collected to extract tolal protein, nuclear protein, and total RNA for western blot, real time-PCR and microarray assays. Thirdly, two non-small cell lung cancer cell lines, NCI-H23 and NCI-H460, were used to explore the relationship between CYP1B1 and ERa with NNK-induced lung cancer carcinogenesis. (a) cell culture: two cell lines were individually cultured in DMED supplemented with 10% FBS at 37?and 5% CO2. According to the different research contents, cells were treated with different chemical including NNK and U0126 or siRNA. (b)Western blot were used to detect the protein expression of ERa and CYP1B1 in cells. (c) Cell apoptosis was analyzed by flow cytometry to evaluate the effect of ERa and CYP1B1 on NNK-induced cell proliferatoin. (d) The expression of ERa and CYP1B1 was measured by western blot assay to identify the locality of ERa and CYP1B1 at signal pathway. (e) RAS/ERK/AP1 signal pathway-related proteins were detected to observe whether RAS/ERK/AP1 signal pathway contributes to NNK-induced lung cancer cell proliferation. These proteins are ERK, p-ERK, c-Fos, c-Jun, pdcd4, spryl, spry2 and btg2. (f)Those proteins were detected after cells treated with U0126 to confirm RAS/ERK/AP1 signal pathway involved in NNK-induced lung cancercell proliferation. Meanwhile, through observing the changes of ERa, CYP1B1, Bax and Bcl-2 protein ensure the impact of ERa and CYP1B1 in NNK-induced cell proliferation. (g) RAS/ERK/AP1 signal pathway-related proteins and apoptosis-related proteins were detected by western blot to discuss whether ERa improving NNK-induced lung cancer cell proliferation depends on CYP1B1 and ERK.(h) Drawing a probable network.Results (1) ERa protein expression in lung cancer tissue was significant more than that of normal tissue which come from the same patient and localed in nucleus. (2) The results of ERa expression assayed by IHC staining of A/J mice lung tissue were consistent with that observed in lung cancer patient's sample. The results of western blot showed that the expression of ERa in nuclear of NNK-treated group was increased comparing to control.And, the level of ERa mRNA in NNK-treated group mice was higher than that of control group, especially at the 30th week and 34th week. (3) The results of western blot showed that CYP1B1 was significantly increased during the progress of NNK-induced A/J mice lung cancer and there was a positive correlation between CYP1B1 and ERa. After 34th week of NNK-treated, microarray showed that the code gene of ERa and CYP1B1 went up 8 fold or 6 fold respectively. Gene network microarray revealed that three gene networks which more than 15 grades included ERa and Cyplbl and were invovled in NNK-induced lung cancer.ERK/MAPK signal pathway was one of the eight signal pathways including ERa and Cyp1b1. (4) (a) In vitro study, ERa and CYP1B1 expression were upregulated in total and nuclear protein of NCI-H23 and NCI-H460. ERa protein expression in lung cancer cells assembled in nucleus too. This result consists with results obtained in A/J mice lung tissue and human lung cancer tissue. (b) The results of flow cytometry showed inhibiting ER? and CYP1B1 could induce cell apoptosis. (c) NNK up-regulated the expression of p-ERK, c-Fos and c-Jun in NCI-H23 and NCI-H460, and c-Fos and c-Jun expression were almost paralleled with p-ERK. However, multiple negative regulatory factors, pdcd4, spryl, spry2 and btg2 were decreased dependently-time with NNK. We found that c-Fos and c-Jun expression were decreased in U0126-treated cells, but the expression of Pdcd4, Spryl, Spry2 and Btg2 was increased comparing to control. (d) U0126 reduced NNK-induced up-regulation of ER?,but improved CYPIB 1 expression. In addition, U0126 increased pro-apoptosis protein, Bax and decreased anti-apoptosis protein, Bcl-2. Above all, we knew blocking ERa by inhibited ERK could cause lung cancer cell apoptosis significantly?Conclusion (I) ERa was advantaged in lung cancer development. (2) Both ER? and CYP1B1 influence in lung cancer carcinogenesis. (3) ERa improves NNK-induced lung cancer carcinogenesis by CYP IB 1 and ERK. (4) Inhibiting CYP1B 1, ERK and ERa maybe slow down lung cancer development and it will be an effective way of lung cancer therapy.