Relationship Between Subpopulation Cells And Cancer Stem Cells In NCI-H446 Lung Cancer Line Cells

BACKGROUND:There is an increasing evidence supporting the cancer stem cell hypothesis.It has been proven that tumor cells are heterogeneous comprising rare tumor initiating cells and abundant nontumor initiating cells.Tumor initiating cells-cancer stem cells have the ability of selfrenewal and proliferation,are resistant to drugs,and express typical markers of stem cells.It is not clear whether cancer stem cells originate from normal stem cells in consequence of genetic and epigenetic changes and/or by redifferentiation from somatic tumor cells to the stem-like cells. Probably both mechanisms are involved in the origin of cancer stem cells. Dysregulation of stem cell self-renewal is a likely requirement for the development of cancer.Isolation and identification of cancer stem cells in human tumors and in tumor cell lines has been successful.To date,the existence of cancer stem cells has been proven in acute and chronic myeloid leukemia,in breast cancer,in brain tumors,in lung cancer and gastrointestinal tumors.Cancer stem cell model is also consistent with some clinical observations.Although standard cheroot-herapy kills most cells in a tumor, cancer stem cells remain viable.Despite the small number of such cells, they might be the cause of tumor reccurrence,sometimes many years after the "successful" treatment of primary tumor.Growth of metastases in distinct areas of body and their cellular heterogeneity might be consequence of cancer stem cell different-tiation and/or dedifferentiation and asymmetric division of cancer stem cells.Further characterization of cancer stem cells is needed in order to find ways to destroy them,which might contribute significantly to the therapeutic management of malignant tumors. Kim and his colleages had isolated such a regional pulmonary stem cell population in 2005,termed bronchioalveolar stem cells(BASCs).Identified at the bronchioalveolar duct junction,BASCs were resistant to bronchiolar and alveolar damage and proliferated during epithelial cell renewal in vivo. BASCs exhibited self-renewal and were multipotent in clonal assays, highlighting their stem cell properties.Furthermore,BASCs expanded in response to oncogenic K-ras in culture and in precursors of lung tumors in vivo.These data support the hypothesis that BASCs are a stem cell population that maintains the bronchiolar Clara cells and alveolar cells of the distal lung and that their transformed counterparts give rise to adenocarcinoma.Although bronchiolar cells and alveolar cells are proposed to be the precursor cells of adenocarcinoma,this work points to BASCs as the putative cells of origin for this subtype of lung cancer.Dong Qiang-gang and his assistants had isolated and characterized the highly tumorigenic cancer stem cells with stem cell feathers from the established human lung adenocarcinoma cell lines in 2008.The cell subtypes were separated by MACS and the stem cell-related markers on these cells were detected.They concluted that the CD 24~+ IGF-1R~+ cells in human lung adenocarcinoma belongs to the BASC-like cancer stem cells.Renewing cancer stem cells exist in Holoclone in A549 lung cancer stem.CD133,ABCG2 maybe the signs on the surface of lung neoplasm cancer stem cells.BMI-1 gene overpressing maybe related with the growth of lung neoplasm cancer stem cells.SP(side population) cells maybe useful for isolation of cancer stem cells in surficant-unkown cells.OBJECTIVE:This study is to investigate the character of heterogeneous clones derived from a single cell,and to explore the mechanism of their heterogeneity;to investigate the expression of BMI-1 in human lung cancer H446(small cell lung cancer,SCLC) line heterogenous subpopulation cells;relationship between them and cancer stem cells in human lung cancer H446(small cell lung cancer,SCLC) line cells;to investigate the Inhibitory effects of sorafenib on NCI-H446 human lung cancer line cells.METHODS:NCI-H446 lung cancer line cells were cultured in vitro, heterogeneous clones were isolated by limiting dilution.CD133 and ABCG2 were detected by immunohistochemistry(IHC) in each clone cells.Cell cycle was examined by flow cytometry(FCM).BMI-1 prote(?) were detected by Western-blotting.The inhibitory effects of sorafenib(?) NCI-H446 human lung cancer line cells were determined by MTT ass(?) after at 24h,48h and 72h after the addition of sorafenib to the NCI-H446 culture. The concentration of sorafenib were 6umol/l,3umol/l,1.5umol/l.RESULTS:15 clones were derived from H446 line cultured cells,they were divided into two sorts termed clone A and clone B morphologyly.The positive rate of CD133 and ABCG2 expression in clone A were(66.67±8.57)%,(33.33±5.17)%;while(15.38±2.68)%,(7.69±1.64)%in clone B(P＜0.05).Furthermore,the ratio of cells in G0/G1 phase were(70.13±3.26) %70.13%and(54.61±1.38)%.The positive rate of BMI-1 protein expression in clone A was 75%while 12.5%in clone B(P＜0.05).Totally,the positive rate of BMI-1 protein expression in H446 line cells was 33.33%. Inhibitory ratio of sorafenib on clone A was 4.96%-6.49%,in contrast, it was about 3.86%-30.39%on clone B.The inhibitory effect of sorafenib on clone B cells could be developed as the time and concentration increased.CONCLUSIONS:Charecters of morphology and tumorigenesis are different in clones,cancer stem cells maybe exist in clone A which over expresses CD133 and ABCG2.BMI-1 is related to tumor progress in different clones,cancer stem cells maybe exist in clone A which over expresses BMI-1.NCI-H446 lung cancer cells may be inhibited by sorafenib, and the effects were increased partly with the time and concentration developed.The different effects on the two clone cells were related to their biological mechanism.