Evaluation of the expression of Notch1 and related proteins in lung carcinoma cells

Introduction Notch signaling pathway has different roles in many human neoplasms, being either tumor-promoting or anti-proliferative. In addition, Notch signaling in carcinogenesis could be tissue dependent.Aim To study the relation between Notch1 protein expression in lung cancer cells to the following Notch related proteins: Hes1, c-Myc, Jagged1 and Jagged2.Materials and methods Notch1 and its related proteins were detected in human lung cancer cell lines and in 54 surgically resected different lung carcinoma tissues. Then, we used small interfering RNA (siRNA) technology, to down-regulate the expression of Notch1 in H69AR and SBC3 small cell lung carcinoma (SCLC) cells. Also, we transfected venus Notch1 intracellular domain (v.NICD) plasmid into human SCLC lines; H69. Result s: The expression of Hes1, c-Myc and Jagged2 is affected by Notch1 in SCLC.Conclusion There is a strong association between the expression of Notch1 protein and the expression of Hes1, c-Myc and Jagged2 proteins, which could aid in better understanding the tumorigenesis in SCLC.


Background
Lung cancer is classi ed into two main types: small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC), which is further sub-divided into: adenocarcinoma (ADC), squamous cell carcinoma (SCC) and large cell carcinoma [1,2]. SCLC accounts for 20% of lung cancer, and is characterized with low survival rates, frequent recurrence and failure of therapy [3].
Notch pathway is one of the most important cell signaling pathways, which acts through the interaction with ligands of the Delta (DLL1, DLL3 and DLL4) and Jagged (Jagged1 and Jagged2) family, leading to the proteolytic cleavage of Notch receptor, releasing the Notch intracellular domain (NICD) into the cytoplasm, which enters the nucleus, and induces the transcription of several genes; Hes1, cyclin D1, cmyc, Akt and others [4].
Notch signaling in tumorigenesis can be either oncogenic or anti-proliferative. In lung carcinoma, we previously showed that Notch1 signaling is suppressed in SCLC, by histone deacetylation around the promoter region of Notch1 [5] and the restoration of Notch1 expression in SCLC leads to the concurrent appearance of epithelial-like areas within the SCLC, and overexpression of Notch1 resulted in inhibition of SCLC growth and could play a role in cell chemo-resistance [5][6][7][8]. Moreover, we showed that in NSCLC, Notch1 expression has a tumor inhibitory effect on ADC cells, but not SCC cells [6]. The present study investigates the possible related proteins to Notch1 signaling, aiming for better understanding of Notch1 pathway in lung carcinoma cells.

Cell lines
Human lung cancer cell lines were purchased from American Type Cell Collection (Rockville, MD): H69, H69AR, H889, and HI668 (SCLC), H358 and H1975 (ADC) and H226 and H2170 (SCC). SBC3 cell line was a gift from Dr.Makato Suzuki (Department of Respiratory Surgery, Graduate School of Medical Sciences, Kumamoto University). A549 ADC cell line was afforded by RIKEN Bio Resource Center (Tsukuba, Japan).
Growth media were purchased from Wako Pure Chemical Industries (Ltd., Osaka, Japan). All cells were cultured as previously described [6].

Western blotting (WB) analysis
Cells were prepared for WB as previously described [6]. List of primary antibodies used are listed in table 1. The membrane was then incubated with the appropriate secondary antibodies (Amersham Pharmacia Biotech, Buckinghamshire, UK), and the immune complex was visualized with the ECL system (Santa Cruz, Texas, US).

Immuno uorescence (IF)
Cells were plated in 24 well plates and were treated as previously described [6]. List of primary antibodies used are listed in table 1. Cells were incubated with the appropriate secondary antibodies (Alexa Flour, Molecular Probes,Eugene, OR) and examined by uorescent microscope (Olympus, Tokyo, Japan).

Histopathological evaluation
Tissue samples of lung ADC (n=31), SCC (n=9) and SCLC (n=14) were obtained from anonymous cases of lung cancer patients, who were surgically treated at Kumamoto University Hospital. All samples were xed in 10% formalin and embedded in para n. Tissue sets were stained with hematoxylin and eosin (H&E) staining and additional sections were used for immunohistochemical (IHC) staining; as previously described [6]. The list of primary antibodies used are listed in table 1. The appropriate secondary antibody (Envision+System-HRP Labelled Polymer, Dako, Glostrup, Denmark). All slides were examined twice, by the researcher and another independent pathologist in a blinded fashion. The localization of Notch1 and its related proteins (NICD, Hes1 and Jagges1) was observed. A semi-quantitative method to assess the staining intensity was used: a strong positive result was de ned as strong immunoreactivity in 50% or more of tumor cells, a weak positive result was de ned as weak immunoreactivity or staining of fewer than 50% of tumor cells, and tumors with no or minimal staining were scored as negative.

Results
Expression of Notch1 and related proteins in lung cancer (Fig.1) By WB, Notch1 and c-Myc were detected in NSCLC cells. In contrast, all SCLC cells-except H69AR and SBC3-lacked Notch1, but weakly expressed c-Myc, as did H1688 cells. Hes1 and Jagged1 were expressed in both SCLC and NSCLC cells (Fig.1a). To detect cellular localization of Notch1 and its related proteins, we performed IF for selected cell lines: H69 and H1688 (SCLC not expressing Notch1), H69AR and SBC3 (SCLC expressing Notch1), A549 and H2170 (NSCLC). In H69 and H1688 cells, Hes1 was detected in mainly in cells nuclei and occasionally in cytosol, while Jagged 1 was seen mainly within cytosol. In the rest of cells expressing Notch1, Hes1 and Jagged1 were detected mainly in cytosol (Fig.1b). To con rm our in vitro ndings, IHC staining of human lung carcinoma tissue was done. In SCLC, Notch1 was absent, Hes1 was weakly positive in nuclei and Jagged1 was strongly positive in nuclei. In NSCLC, Notch1 and NICD were strongly positive in ADC, while weakly positive in SCC. Hes1 was weakly expressed mainly in cytoplasm of cells, while Jagged1 was strongly positive mainly in cytosol of cells (Fig.1c).
Knocking down (KD) Notch1 and transfection of Notch1 venus NICD (v.NICD) plasmid WB, IFA and RT-PCR were used to detect the e cacy of siRNA against Notch1 in transfected cells and to ensure v.NICD transfection into H69 cells (Fig.2).
Effect of Notch1 KD and overexpression on Notch related proteins (Hes1, c-Myc, Jagged1 and Jagged2) ( Fig. 2) In SCLC, Hes1 and c-Myc protein expressions were decreased in cells with KD Notch1 and increased in H69 cells transfected with v.NICD plasmid. In NSCLC, neither Hes1 nor c-Myc protein expressions were affected by KD Notch1. Regarding Jagged1, its expression wasn't affected by either KD or induction of Notch1. However, Jagged2 expression was increased in SCLC cells with KD Notch1; especially H69AR cells and in H69 cells transfected with v.NICD. Moreover, Jagged2 expression was decreased in NSCLC cells with KD Notch1, especially A549 cells.

Discussion
Despite rapidly accumulating information, the role of Notch signaling in oncogenesis is far from fully understood, due to the complex nature of Notch signaling and that differences in cell type could affect its nal outcome [9]. Our present report focuses on the relation between Notch1 and Notch related proteins in SCLC and NSCLC cells.
Our data showed that Notch1 receptor and its related proteins (Hes1, c-myc and Jagged1) were signi cantly overexpressed in NSCLC and not in SCLC, consistent with other's observations [10][11][12]. In SCLC, we detected also expression of Hes1 and Jagged1 proteins despite the absence of Notch expression, while no expression of c-Myc protein was detected, except in H69AR and SBC-3-both expressing Notch1-and H1688 cells, which don't express Notch1. Such Hes1 protein expression in SCLC that do not express Notch 1 can be explained by the fact that other signaling pathways control Hes1 protein expression, as EGFR and FGFR [13]. Regarding c-Myc, its expression in SCLC cell lines has been reported in the variant class of SCLC; that is characterized by adherent cell growth and morphology similar to undifferentiated LCC [14,15]. In our study, we used the following SCLC cells: H69, H889, H69AR, In addition, we demonstrated that Hes1 and Jagged1 were detected in the nuclei of SCLC cells. There may be some undetected mechanisms for Hes1 to go inside the nuclei from the cytosol, as suggested by previous studies [19,20]. On the other hand, nuclear localization of Jagged1 can be explained by the fact Jagged1 has an intra-cellular domain, which contains nuclear localization signals that permit their entry into the nucleus [21]. Regarding NSCLC, cellular localization of both Hes1 and Jagged1 show differences between detecting them in cell lines compared to tissue sections. In cell lines, both proteins were seen in cytosol of NSCLC cells. In tissue sections, both proteins were detected in cytosol and nuclei of ADC cells, while they were mainly seen in nuclei of SCC cells. This could be attributes to the antibodies used or to the difference in cell biological behavior when grown in cell lines or in tissues. We believe that clarifying the mechanism of Notch1 related proteins subcellular tra cking may give an additional insight for better understanding the role of Notch1 signaling in lung carcinoma.
By utilizing siRNA analysis, we demonstrated the effect of KD of Notch1 in H69AR and SBC-3 cells and con rmed such effect by observing the results of overexpressing Notch1 in H69 cells. Moreover, we showed the effect of KD Notch1 in NSCLC cells; A549 and H2170 cells.
We found that KD Notch1 decreased Hes1 and c-Myc expression in transfected H69AR and SBC-3 cells, and that expression of Notch1 in H69 cells increased their expression. This indicates close interaction between Notch1 with Hes1 in SCLC, as previously reported [22,23]. In addition, our results suggest that c-Myc is a downstream molecule of Notch1 signaling in SCLC cells, as previously reported in T cell acute lymphoblastic leukemia [24][25][26]. In NSCLC cells, we couldn't observe any effect of KD Notch1 on Hes1 or c-Myc expressions, suggesting that these two proteins are not related to Notch1 signaling in NSCLC cells. Moreover, we found that Notch1 affect Jagged2 expression-in both SCLC and NSCLC cells-while has no effect on the expression of Jagged1. This can be explained by the fact that Jagged1 is associated with Notch3 signaling in lung carcinoma-as we previously reported- [27], and in other carcinomas; ovarian carcinoma, pancreatic cancer and cervical SCC [28][29][30][31]. Moreover, our results con rm the fact that Jagged1 and Jagged2 have different biological roles as previously stated [32].
In comparison to Notch3 signaling, we showed that -in both NSCLC and SCLC cells-Jagged1 and Hes1 expressions were affected by Notch3 protein, and that Nocth1 protein expression was decreased by KD Notch3 in H69AR cells, indicating a close interaction between both Notch1 and Notch3 signaling in SCLC cells [27].