| Lung cancer is the most common cause of cancer-related mortality and its incidence has been steadily rising worldwide over the past 20 years.Lung cancer is divided into small cell lung cancer(SCLC)and non-SCLC(NSCLC).The most common forms of NSCLC include adenocarcinoma(AC),squamous cell carcinoma(SCC)and large cell carcinoma(LCC),which comprises about 80% of lung cancer.Patients with lung cancer frequently present with metastases regardless of the primary tumor size at the initial diagnosis and always have a high rate of relapse after treatment.Therapeutic approaches for lung cancer are multifactorial,include surgery,immunotherapy,radiotherapy and targeted therapy.Despite the widespread use of multimodal treatment,the overall five-year survival rate for such tumors is lower than 15%.Recent advance revealed that a lot of malignant tumors,including lung cancer,are composed of diverse cell types with distinct proliferative and differential capacities.Thus,the emerging reasonable explanation is the existence of a rare subpopulation of cells which are purported cancer stem cells(CSCs)that may be contribute to some cases of resistance to cancer therapy and relapse.In fact,several researchers suggest that a stem like subpopulation derived from lung cancer cell lines and tumor specimens has been isolated by flow cytometry according to detection of side-population(SP)phenotypes.It was reported that NSCLC(H460,H23,HTB-58,A549,H441,and H2170)cell lines contained SP cells ranging from 1.5% to 6.1% of the total viable cell population.In another study,SCLC cell lines(H146 and H526)were found to comprise 0.7 to 1.3% of SP cells,while NSCLC cell lines A549 and H460 contained 2.59% and 4.00% of SP cells,respectively.Strikingly,NSCLC cell line A549 used in the above-mentioned studies showed a significantly different SP fraction ranging from 2.59 to 24.44%.Those results indicate that the frequency of SP fraction appears to be highly variable between different lung cancer cell lines and among same type.This issue may be related to the use of lung cancer sublines passaged for different generations in individual laboratories.Emerging evidence revealed that repeated passaging of cell lines for many generations frequently leads to change of characteristics,such as alterations in cell morphology,growth rates,protein expression,and cell signaling,and the acquisition of genetic aberrations.Actually,the used cancer cells have been passaged many times within one laboratory.Based on these findings,it is worth investigating the effects of repeated passaging on the biological and functional properties of the enriched SP fraction from early and late passage cells.In order to test this hypothesis,A549 and NSCLC SP cells from low passage and long-term passage cells was isolated by utilizing flow cytometry base on ABCG2 efflux pump-mediated Hoechst 33342 dye exclusion.The isolated SP cells were used to investigate whether increasing cell passage could alter their CSCs-associated biological and functional properties.This could be helpful for explaining current blurred results and better understating the biology of NSCLC CSCs.Part I Sorting of side population in NSCLC cell lines passaged for different generationsMethods1.To sort the side population(SP)cells in A549 NSCLC cell lines and primary NSCLC cells passaged for different generations(2nd and 50th).2.To identify the SP fraction by utilizing flow cytometry process in the presence of verapamil.Results 1.A549 cell lines contained a similar fraction of SP cells between 2nd(2.78%)and 50th(2.03%)passage cells.For early and late passage NSCLC cells,SP cells accounting for 5.81% and 4.93% of the whole population,respectively.2.The SP fraction decreased significantly in the presence of verapamil.Conclusions 1.Isolation of SP fraction from different passage A549 and NSCLC cells.2.The SP fraction from A549 and NSCLC cells decreased after long term passage.Part II Identification of cancer stem cell properties in side population from NSCLC cell lines passaged for different generationsMethods 1.To determine differences in clonogenic capacity of SP fraction from 2nd and 50 th passage cells,the clone formation assay were performed.2.Sphere formation has been recognized as a defined characteristic of CSCs that reflects the potential of self-renewal ability.Thus,the sphere formation capacity was evaluated.3.Based on the notion that CSCs frequently have conserved stem and progenitor cell phenotypes,the expression of embryonic stem cell(ES)associated genes were evaluated.4.Serial transplantation is the the most widely accepted assay and recommended to determine stem cell properties.Thus,the tumorigenic potential of SP subpopulation within A549 and NSCLC cells were investigated.5.The tumor cell migration associated genes were also evaluated.The migration and invasion abilities of CSCs were frequently associated with epithelial-mesenchymal transition(EMT).Thus,EMT process associated genes such as E-cadherin,N-cadherin,snail and twist were investigated.Results1.The results showed that the mean clone formation efficiency was 79.81±5.71% and 66.84±3.47%(P<0.05)in early and late passage A549 cells,respectively.For NSCLC cells,the percentage of formed clones in 2nd and 50 th passage cells was 78.27±2.26% and 61.45±3.72%(P<0.05),respectively.2.The sphere formation efficiency of 2nd and 5th passage sublines were 15.70±2.42% and 9.45±0.68%(P<0.05)in A549 cells,and 12.46±1.63% and 8.08±1.50%(P<0.05)in NSCLC cells,respectively.3.The real time PCR analysis revealed that the m RNA expression of 2nd passage SP cells(except Bmi-1 expression in A549 cells)has no significantly differences as compared to 50 th passage SP cells.4.The results observed that tumor-initiating frequency was decreased after long term passage.Subcutaneous tumor formation required at Ieast 1 × 104 SP cells from early passage A549 cell lines,or 1×105 SP cells from late passage A549 cell lines.The same event required at Ieast 1 × 102 SP cells from early passage NSCLC cell lines,or 1×104 SP cells from late passage NSCLC cell lines.This result was also confirmed by micro PET imaging.5.Real-time PCR analysis indicated that the genes of N-cadherin,snail and twist were expressed higher in 2nd passage cells as compared to 50 th cells(P<0.05).For the E-cadherin m RNA expression,which is increased in 50 th A549 cells while has no significantly difference in NSCLC cells.Conclusions 1.The stem cell like properties of SP fraction from A549 and NSCLC cells were diminished after long term passage.2.Primary cancer cells or low passage cell lines were recommended in CSCs field.Part III Study of Chemoradiotherapy Resistance in side population from NSCLC cell lines passaged for different generationsMethods1.To determine whether long term passage have effects on sensitivity of SP subpopulation to irradiation by application of single-hit multi-target model,the values of N,D0,Dq and of 2nd and 50 th passage SP cells were analyzed.2.To determine whether long term passage have effects on sensitivity of SP subpopulation to chemotherapy,the cytotoxicity of doxorubicin on different passage A549 and NSCLC SP cells were evaluated.3.Doxorubicin and ionizing radiation induced apoptosis in early and late passage cells were analyzed.Results1.The results indicated that 2nd passage cells exhibited more resistance than 50 th passage cells in A549 and NSCLC SP cells.The survival fraction after 2 Gy irradiation were 55.84%±1.91% for 2nd passage cells vs.50.26%±2.24% for 50 th passage cells in A549 cell line,and 70.32%±1.52% for 2nd passage cells vs.58.13%±1.21% for 50 th passage cells in NSCLC cells.2nd passage SP cells were more resistant to irradiation as compared to 50 th passage SP cells.2nd passage cells display significantly larger values of Dq than 50 th passage cells,indicating that the repair of sublethal damage capacity was diminished with increasing cell passage.2.The SRB assay indicated that 2nd passage subline were more resistent to high dose of doxorubicin(1000 n M)as compared to 50 th passage subline in A549 cells.For NSCLC cells,50 th passage subline showed more sensitivity to high dose of doxorubicin(1000 n M)as compared to 2nd passage subline,while has no significant difference on low dose of doxorubicin.Additionally,the A549 cells exhibit no significant different in sensitivity to the low dose of doxorubicin.3.The apoptotic cell death rate was 7.94±0.82% and 10.32±1.13%(P<0.05)in early and late passage cells after 2 Gy irradiation,respectively.After 500 nm doxorubicin pre-incubation,the apoptosis rate in 2nd and 50 th passage cells was 22.76±2.81% and 27.95±3.17%(P<0.05),respectively.The similar results were observed in low and long term passage NSCLC cells,the apoptotic cell death rate was increased in 50 th passage cells(P<0.05).Conclusions1.SP fraction derived from different passage cells display distinct chemo-and radio-sensitivity.2.SP fraction from NSCLC lines exhibited less resistance to chemotherapy after late passage. |
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