Expressions And Correlations Of Connexin43 And E-cadherin In Lung Cancer

Objectives and significanceSince 1960s Loewenstiein and Kanno discovered connection among cancer cells was loose and even lost, many studies revealed the gap junction play a critical role in progression of tumorigenesis development. The deficiency of gap junction communication is widespread in cancer cells. With the development of relative techniques, we further revealed the gap junction communication contribute to progression of decancerate , renewed gap junction communication will reverse the malignant phenotype or suppress growth of tumor cell. Thus, the study of gap junction has came to a new hotspot of oncobiology.Gap junction is a kind of membrane channel, which is constituted of trans-memb - rane Connexin (Cx). Through gap junction, cells transmit signals to each other and average metabolites. By now, functions of gap junction are as below : (1) it participates varing signaling pathway; (2) it contributes to fetal development ; (3) it takes part in adhesion and movement of cells; (4)it has a hand in local metabolism; (5 ) it buffers toxicant. (6 ) it nourishes impaired cells via neighboring cells. Reduced expression of connexin will cause descent of communication among cells, and then lead to canceration.The protein component in the gap junction is called Connexin (Cx) , varing Cx are coded by genes on different chromosomes. Cx gene is a multiple gene family, containing many homologous genes. More than ten members in the family were discovered by now. cDNA colonal sequencing analysis showed they had high homology and several highly conserved sequences. All the Cx genes have two exons: one is short, the other has a intact encoding sequence. But introns have long but varying length. Cx has four hydrophobic transmembrane regions,conforming a " M" model molecular chain, the amino - terminal and carboxy -terminal are in intracellular.As widely reported, reduced expression of Cx gene was widespread in tumors, then lead to weak or deficient function of gap junction communication. It suggested that deficiency of gap junction communication was a common event in canceration and tumorigenesis. After transfecting Cx cDNA, tumor cells will renew functional gap junction communication and suppress proliferation of tumor cells, even refresh the potential differentiation capability and achieve malignant transformation reversal.E - cadherin, a transmembrane glycoprotein of the type -1 cadherin super-family, encoded by the gene on chromosome 16q22. 1. Its cytoplasmic part is linked to the actin cytoskeleton via the catenins, α - , β - , γ - catenin (the same as plakogobin). The extracellular part of E - cadherin mediates cell - cell and cell - extracellular matrix adhesion. It is by now quite clear that E - cadherin is not only a cell - cell adhesion molecule but is also pivotal in signal transduction from the outer cell surface to the cytoskeleton. In differentiated cells, the minus ends of microtubules are stabilized by signaling from cadherins through cell - cell contact. Connections between the E - cadhein/Catenin complex and multiple other receptor and nonreceptor signal - transducing systems may contribute to the complexity of the cancer phenotypes.Both Cx and E - cadherin are transmembrane glycoprotein, their synergism mediates gap junction. E - cadherin mediates conforming Ca2 + - dependent gap junction and facilitates Connexin transporting from cytoplasmic to cell membrane. Both Cx and E - cadherin have association to tumorigenesis and development. But it is still unclear whether there is relationship between expression of E - cadherin and Cx, and exist interaction between them. Thus, we detected expression of Cx43 and E - cadherin by immunohistochemistry in pulmonary primitive squamous cell carcinoma and adenocarcinoma which had been determined by pathological diagnosis; carried out analysis of relationship between expression of E - cadherin and Cx; transfected the Cx43 gene to pulmonary giant cell carcinoma PG cell lines, and detected expression of Cx43 and E - cadherin in LH7 cell and LH7Cell were stably transfected with a vector that constitutively ex-pressed Cx43 gene, then analyzed the effect of transfecting Cx43 to expression of E - cadherin. The aim of the present study was to provide a framework for further investigating the mechanism of Cx43 and E - cadherin in tumorigenesis as well as development.MethodsImmunohistochemistryTo investigate expression of Cx43 and E - cadherin in 85 pulmonary primary squamous cell carcinoma and adenocarcinoma, we performed immunohistochemistry, then analyzed the correlation between them.Expression of Cx43 and E - cadherin determine1. Origin of vector; the vector for Cx43 gene was pcDNA3.1 ( + ) - Cx43 , recombinated by TAKARA Inc, a gift of prof. Youwei Zhang, University of Tokyo Medical and Dental. The Cx43 gene is a 1. 367kb fragment , amplified by Bacterium coli,then isolated using Plasmid kit(QIAGEN Inc. )2. Cell cultue: human high metastasis pulmonary giant cell carcinoma PG cell lines subseries of BEj and LH7 a gift of Beijing Medical University. BEj and LH7were cultured in RPMI1640 medium(GiBCO Inc. ) with 15% fetal calf ser-um(GiBC0 Inc. ) , 2g/L NaHC02, 100U/L penicillin - G, lOOfxg/mL, CO2, 37°C.3. Transfection LH7 and screening colon cell: LH7cells were transfected into pcDNA3.1( + ) ^pcDNA3.1( + ) -Cx43 using FuGENE 6(Roche) and selected antibiotics G418( Roche) by plasmid with neomycin resistance.4. Western blot; To detected expression of Cx43 and E - cadherin in LH7 cell and LH7 Cell were stably transfected with a vector that constitutively expressed Cx43, we performed Western blot. All cells including LH7Cell were stably transfected with empty vector ( - ) or with a vector that constitutively expressed Cx43 gene were lysed in 200ui RIPA buffer, Coomassie brilliant blue quantitative determinated protein; polyacrylamide gel electrophoresis; After 2 hours of transfering to PVDF membrane, the blot was probed with murine poly-clonal anti -Cx43 antibody (1:300,SantaCruz, CA) ^murine polyclonal anti -E - cadherin antibody ( 1:300, SantaCruz, CA) ^ murine polyclonal anti - (3 -catenin antibody (1:300,Santa Cruz,CA). The secondary antibody was murine polyclonal anti - alkaline phosphatase, then incubated 2 hours in room temperature and stained with alkaline phosphatase (Sigma) 5 min. For quantification, the bands were analyzed with FluorChen V. 2.0 system( Chemilmager 5500, Al-Pha InnCh) , Density value of bands were served as index of intensity, used p -actin as internal reference, all the density value of bands were compared with |3 - actin. Thus 3 times and used arithmetical mean for later statistical analysis.5. Immunofluorescence: LH7 and LH7Gjal were inoculated in 24 pores plate with density of 2 x lOVml at the same time, after 48 hours fixed in 95% alcohol for 10 minutes; and penetrated with 0. l%TritonX -100 for 10 minutes; incubated with 2% BSA for 1 hour; respectively added murine polyclonal anti - Cx43 antibody(1:300,SantaCruz, CA) and murine polyclonal anti - E - cadherin antibody (1:300,SantaCruz, CA) for overnight at 4 °C; respectively labeled with murine anti - FiTC fluorescein as secondary antibody, incubated for 1 hour at 37 °C; added 0.1% DAPI for karyotin for 3 minutes; rinsed in PBS + Triton( 10: 1) and then in PBS. Imaged by Immunofluorescence microscope ( BX60, 0-LYMPUS). When the cells had FITC fluorescein, it was judged to positive for expression.Determine growth rate and cell cycle of transfected cells:1. Determine growth rate of transfected cells: To determine growth rate of trandfected cells, we performed Celltlter 96? AQueous ( Promega, Madison, WI). LH7and LH7Gjal were inoculated in 96 pores plate with density of 500/ pore at the same time, then added lOOpi RPMI 1640 containing 15% fetal calf serum. Each type of cells was inoculated in 24 pores (4 lines x 6 arries). The 4 lines were 4 parallel samples, the 6 arries were performed ELISA every day. Before determining, we added 20jjJ MTS/PMS(Promega) in each pores, then incubated with madder air containing 5% C02 at 37°C. After 3 hours of incubation , we measured the absorbance of each pore, used arithmetical mean of 4 parallel samples for later statistical analysis.2. Determine cell cycle of transfected cells; LH7 and LH7Gjal were inoculated in pore plate with density of 1. 5 X lOVml at the same time. Collecting cellsto 10ml centrifuge tube in one time after culturing 48 hours, then centrifuged at 1000G for 5 minutes at 4°C; discarded supernatant and added lml cold PBS buffer and 200jxg/ml RNase in each tube ,then inoculated for 30 minutes at 37° C;at last , added lmg/ml Propidium iodide (PI, Sigma) and incubated for 10 minutes at room temperature; caught data by flow cytometer (FACSCalibur, BD America). The index of comparing LH7 and LH7 Gjal was percentage of Gj phase ^G2 phase and S phase in all cells.Statistical analysis: The X2 test and spearman correlation analysis were used to clarify the relation between the result of immunohistochemisty; The result of Western blot >, cell growth rate N cell cycle were analyzed by t - test.ResultImmunohistochemistry:15 cases showed typical membranous expression of Cx43 and E - cadherin in normal pulmonary tissue, suggesting there were normal gap junction and cell adhesion among cells. However, only 43 cases showed positive expression of Cx43 in 85 cases of pulmonary carcinoma tissues, accounting for 50. 6%. 57 cases showed E - cadherin positive expression, accounting for 67. 1%. The expressed intensity of Cx43 and E - cadherin reduced obviously, both of them were mainly expressed in cytoplasm, a few of them were expressed in nucleus or/and membrane; There were significant differences in Cx43 and E - cadherin expressions between patients with degrees of differentiation ? different P -TNM classifications and lymph node metastasis; Namely, with the descendent degrees of differentiation, the rate of positive expression descended obviously. For example, there was no expression of Cx and E - cadherin, especially the Cx, in some tissues of poor differentiation. The positive rates of Cx and E - cadherin were rising with the descendent degrees of differentiation; in tissues with lymph node metastasis, the positive rates of Cx43 and E - cadherin were lower than tissues with no lymph node metastasis. Both Cx and E - cadherin had no significant difference in tissue types.Expression of Cx43 and E - cadherin determineAs reported, expression of Cx43 in BEt and LH7was lower than normal cells. However, our results of western blot shown that the expression of LH7 was lower than BE,.After LH7was trandfected the gene of Cx43, results of western blot showed that the density value of Cx43 bands were higher than LH7 cell and LH7Cell were stably transfected with empty vector. Band intensity was quantified using densi-tometry and was normalized to p - actin band intensity. There was significant difference between LH7 cell and LH7Cell were stably transfected with a vector that constitutively expressed Cx43.Similarly, after LH7 was trandfected the gene of Cx43, results of western blot also showed that the density value of E - cadherin bands were higher than LH7 cell ,the data were standardized by |3 -actin. There was significant difference between LH7 cell and LH7Cell were stably transfected with a vector that constitutively expressed Cx43.After LH7was trandfected the gene of Cx43, results of Immunofluorescence showed that expression of Cx43 was weak LH7 cell, and strongly expressed in LH7Cell were stably transfected with a vector that constitutively expressed Cx43. No matter transfection or no transfection, the expression of Cx43 was mainly in cytoplasm but none on membrane. Namely, the expression of Cx43 was enhanced in LH7Cell were stably transfected with a vector that constitutively expressed Cx43, but no gap junction was established among LH7cell.After LH7was trandfected the gene of Cx43, results of Immunofluorescence showed that expression of E - cadherin was weak LH7 cell, and strongly expressed in LH7Cell were stably transfected with a vector that constitutively expressed Cx43. No matter transfection or no transfection, the expression of E -cadherin was mainly in cytoplasm but none on membrane.Determine growth rate and cell cycle of transfected cells:The average growth rates of LH7 Cell were stably transfected with a vector that constitutively expressed Cx43 gene was obviously lower than LH7 cell. The proportion of Gj phase cells were higher and proportion of S^G2 phases were lower than transfection. However, it is still unclear that there was no significant difference in average growth rate ^proportion of Gj,^ and S phases in some LH7cells.Conclusion1. In pulmonary squamous cell carcinoma and adenocarcinoma, expression of Cx43 was linked to expression of E - cadherin.2. After pulmonary giant cell carcinoma PG cell lines subseries of LH7 was transfected, expression of E - cadherin was associated with expression of Cx43.3. After LH7was trandfected the gene of Cx43, there was no evident membrane expression, that is, no gap junction was established. But the proportion of Gx phase cells were higher and proportion of SNG2 phases were lower, that is , the cell growth velocity was refrained, suggesting growth cycle of LH7cells were altered by transfection. In present study, hold — back of the cell growth velocity was not due to gap junction communication, but was associated with regulatory mechanism of cells.