Characterization Of Side Population Cells From Laryngeal Cancer Cell Lines

PARTⅠIdentification and sorting side population cells in laryngeal cancer cell lines Hep-2 and AMC-HN-8Objectives To investigate the existence of side population cells in laryngeal cancer cell lines and establish the protocol of identification and sorting.Methods Laryngeal cancer cell lines Hep-2 and AMC-HN-8 were used, and the final concentration of Hoechst was 5μg/ml, Verapamil was 150μmol/L, and PI 1μg/ml, Flow cytometry and fluorescence-activated cell sorting (FACS) were performed to identify and sort SP and non-SP, the purity of SP and non-SP was examined after sorting. SP and non-SP were planted in RPMI 1640 with 10% FBS to observe the cell viability and morphology after the culture.Results Side population cells were exist in laryngeal cancer cell lines Hep-2 and AMC-HN-8, and the proportion was 17.1±2.0% and 11.8±1.7% respectively, the purity rate of side population from Hep-2 cell line was 99.2±0.2%, and non-SP was 98.5±0.5% after sorting; Dead cells were few after SP and non-SP cells were planted in RPMI1640 with 10% FBS, they all showed typical characteristics of squamous carcinoma.Conclusions SP cells do exist in laryngeal cancer cell lines and could be inhibited by the inhibitor of ATP-binding cassette (ABC) transporters, verapamil; FACS could sort SP cell effectively, and the toxicity of Hoechst 33342 is limited. PARTⅡCharacterization of cancer stem cell-like side population from laryngeal cancer cell line Hep-2Objective To explore the characterization of cancer stem cell-like side population from laryngeal cancer cell line Hep-2.Methods 1×104 SP and non-SP cells were planted in serum free RPMI1640 in 96-well cell culture plate, quantification of viable cells through reading of UV absorption spectrums at 450 nm was performed on a Versamax microplate reader using CCK8 method on the day 1,3,5,7 days after planting, and the proliferative curve were drawn to compare proliferative capacity of the two groups of cells; The SP rate were examined on the day 0,4,8,12 days after SP and non-SP cell were planted in RPMI 1640 with 10% FBS to compare their differentiative ability, average data of different groups were used to draw curves of differentiation; The cells were treated by radiotherapy by 2 Gy on the 1 day after 1×104 SP cells and non-SP cells were planted in RPMI1640 10% FBS in 96-well cell culture plate, and 2 days later, the absorbance rates using CCK8 method were examined to test their sensibility; The cell cycle status of sorted and unsorted cancer cells were also compared by flow cytometry. To compare the tumorigenic potential,1×105,5×104 and 2×104 SP and non-SP cells were injected subcutaneously into the subcutaneous space of the axillary fossa of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice, tumors grown in mice were counted and immediately removed and stained with HE. Positive ratio of two groups was analyzed by Statistical analysis.Results SP and non-SP cells grown in serum free RPMI 1640 medium (supplemented with bFGF, EGF and insulin) were floating, single, spherical, and lucent. As days passed, nonadherent single SP cells became clusters. Seven days after culture, the size of cell clusters became large because of conglomeration, non-SP cells could not form cell clusters. The absorbance of the cells using the CCK-8 method of side population on the day 1,3,5,and 7 days were 0.665±0.017,1.086±0.069, 1.387±1.107 and 1.675±0.07, respectively, but non-SP cell were 0.694±0.053,0.951±0.031,1.049±0.092 and 1.008±0.086, respectively. They showed significant difference except the first day. The percentage of SP cells decreased in culture over time,47.8±1.1% on day 4,27.8±3.6% on day 8, and 17.3±1.9% on day 12. On day 12 of culture, the percentage of SP cells was similar to that of unsorted Hep-2 cells. However, we also found that non-SP populations generated SP and non-SP, with SP comprising 2.2±0.1% on day 4,4.7±0.4% on day 8, and 4.9±0.3% on day 12. After radiation treatment, the final inhibition rate of SP was 6.7±3.5% and that of non-SP was 29.9±4.9%, respectively, which was a statistically significant difference (t= 14.295,p=.000). Comparison of the cell cycle status of sorted and unsorted cancer cells after sorting revealed that both cells exhibited a similar cell cycle distribution. With the lowest inoculation cell number (2×104) SP cells formed 6 tumors, whereas non-SP cells formed none in mice (p=.007). When the inoculation number was 5×104, SP and non-SP cells formed 7 and 2 tumors (p=.041), respectively. Pathology results confirmed that the tumors formed by SP and non-SP cells were typical of human laryngeal cancer cells.Conclusions Side population (SP) cells purified from Hep-2 cell line harbor cancer stem cell-like properties, showed characteristics of self-renewal, high proliferative capacity, remarkable radiotherapy resistance, and strong tumorigenic ability, and may be related to the radiotherapy resistance of laryngeal cancer. The population of cells is however, heterogeneous, indicating that SP cells are not identical with stem cells. PARTⅢAnalysis of stem cell related genes in side population cells from laryngeal cancer cellsObjective To explore the expression difference of the stem cell related genes CD133, ABCG2, Bmi1, Notch2 and PTEN between SP and non-SP cells.Methods CD133, ABCG2, Bmi1, Notch2 and PTEN of SP and non-SP cells were detected using Realtime PCR.Results CD133, ABCG2, Bmi1and NOTCH2 had higher expression in SP cells, and no difference was existed in the expression of PTEN between SP and non-SP cells.Conclusions Stem cell related genes may play an important role in the formation and maintainance of SP cells in Hep-2 cells; These genes may be the potential therapeutic targets for the treatment of laryngeal cancer.