| Objective: Hexavalent chromium is an important human carcinogen associated with lung cancer and other pulmonary diseases.Exposure to hexavalent chromium induces DNA damage and malignant transformation in human lung epithelial cells.Despite extensive studies,the molecular mechanisms remain elusive,it is also not known whether hexavalent chromium-induced transformation accompany with invasive properties to facilitate metastasis.Therefore,this study is designed to establish a malignant transformation cell model of human bronchial epithelial cells Beas-2B by hexavalent chromium in vitro and to investigate: 1.The effects of hexavalent chromium on proliferative activity of Beas-2B cells;2.The effects of short-term hexavalent chromium induction on migration,invasion and the related proteins in Beas-2B cells;3.The effects of chronic induction of hexavalent chromium on migration,invasion and the related proteins in Beas-2B cells;4.Whether chronic induction of Beas-2B cells by hexavalent chromium induces cell malignant transformation;5.The role of tumor suppressor gene LKB1 on migration and invasion in stable hexavalent chromium-transformed cells Beas-2B-Cr.Then we further clarify the carcinogenic mechanism of hexavalent chromium in cellular and molecular levels and reveal the mechanism of migration and invasion during hexavalent chromium induced cell malignant transformation.We focus on the role of LKB1 in cell malignant transformation and establish a better model to investigate the molecular mechanism of heavy metal induced carcinogenesis,and also provide a theoretical and scientific support for detecting the carcinogenicity of chemical substances and prevention and therapy of occupational lung cancer.Methods: 1.Human bronchial epithelial cells Beas-2B were treated with different concentration of potassium dichromate?K2Cr2O7?solution?0-20?M;0-5?M;0-1?M?for 6?12?24?48 and 72 h,MTT assay and colony formation assay were used to assess the effects of hexavalent chromium on cell proliferative viability,and screen the concentration of hexavalent chromium for long-term induction;2.Cells were pretreated with low concentration?0.125?M ? 0.25?M?of potassium dichromate?K2Cr2O7?for 24 h and 48 h,the effects of chromium on the migration and invasion in Beas-2B cells were detected by Transwell assay.The expression of LKB1 and the related proteins of migration and invasion as follows FAK,Src,MMP-2,GSK3?,?-catenin and HEF1 were analyzed by Western blot;3.Soft agar colony formation assay was used to analyse the malignant transformation of Beas-2B cells by chronic induction of hexavalent chromium?0.125?M?0.25?M?for 20?40 and 60 generations.Transwell assay and Western blot were used to detect the effects of hexavalent chromium on cell migration,invasion and the expression of related proteins during malignant transformation;4.Soft agar colony formation asasy and tumor formation in nude mice were used to detect the ability for tumorgenesis of hexavalent chromium-transformed cells Beas-2B-Cr.The differential expressed proteins in chromium-transformed cells Beas-2B-Cr were analysed by proteomics assays;5.The capacities of migration and invasion and the expression of related proteins in chromium-transformed cells Beas-2B-Cr were assessed by Transwell assay and Western blot;6.Overexpression LKB1 by plasmid STK11 transfection and silencing LKB1 expression by siRNA gene interference were used to analyze the roles of LKB1 on cell migration,invasion and the relationships between LKB1 and FAK,Src,MMP-2,GSK3?,?-catenin,HEF1 and other proteins.Results: 1.High concentration of hexavalent chromium compounds?5-20?M?had a greater effect on the proliferative viability of Beas-2B cells in a time-and concentration-dependent manner?P<0.05?.In order to eliminate the interference caused by the inhibition of proliferation,low concentrations?0.125?M and 0.25?M?of chromium were used as long-term induction and the acting concentration in migration and invasion assays;2.The results of Transwell assay showed that hexavalent chromium compounds could promote cell migration?P<0.01?for 24 h and 48 h short-term treatment,and the ability of cell migration increased in a concentration-dependent manner.The results of Western blot showed that LKB1 decreased with the increase of hexavalent chromium concentration,while FAK,Src,MMP-2,GSK3?,?-catenin and HEF1 were on the contrary;3.The cells induced by hexavalent chromium for 2 months could form colonies in soft agar,and obtained anchorage independent growth ability in a concentration-dependent manner?P <0.05?.The number and size of colonies increased with the induction time of hexavalent chromium.In the chronic transformation,hexavalent chromium frequently induced the migration and invasion of Beas-2B cells in a concentration-dependent manner.The expression of LKB1 was consistently inhibited during cell transformation,whereas the proteins related to migration and invasion were elevated;4.Chromium-transformed cells could form colonies in soft agar and tumors in nude mice.The migrative and invasive ability of the chromium-transformed cells were significantly enhanced compared with the control group?P <0.001?.The expression of LKB1 in the cells was evidently lower than that in the control group,while the expression of proteins related to migration and invasion were higher compared to control group;5.2D-DIGE assay revealed that there were 159 differentially expressed protein spots in chromium-transformed cells compared with the control.The mass spectrometry analysis confirmed that 35 differentially expressed proteins including down-regulated tumor anti-oncogene LKB1;6.In Beas-2B-Cr cells,overexpression of LKB1 could suppress the migration and invasion of the cells,while decrease the expression of FAK,Src,MMP-2,GSK3?,?-catenin and HEF1;Silencing LKB1 by siRNA could promote cell migrative and invasive ability,while increase the expression of FAK,Src,MMP-2,GSK3?,?-catenin and HEF1.Conclusions: The inhibitory effect of hexavalent chromium on the proliferative viability of Beas-2B cells is in a time-and concentration-dependent manner.Chromium-transformed cells can form colonies in soft agar,and get the characteristics similar to cancer cells;Chromium-transformed cells induce tumorigenesis in xenograft assay,and the cells were tumorigenic.Low concentration of hexavalent chromium can induce the malignant transformation of Beas-2B cells;Long-term exposure of hexavalent chromium can induce the migrative and invasive ability of Beas-2B cells and inhibit the expression of LKB1 while activate proteins relating to migration and invasion in the cells.LKB1 regulates the downstream proteins such as FAK,Src,MMP-2,GSK3?,?-catenin and HEF1,and then affects the migration and invasion of Beas-2B-Cr cells. |
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