ABSTRACT
Objective To investigate the effect of curcumin an extract of a Chinese medical herb on the sensitivity of CD133 + rectal cancer cells to radiotherapy.Methods In vitro experiments:CD133 +cells were purified with immunomagnetic beads from HRT-18 cell line and divided into curcumin group,radiotherapy group and curcumin plus radiotherapy group.MTT assay and Annexin V/PI staining were used to measure the proliferation and apoptosis of the cells.In vivo experiments:Transplanted rectal tumor was established in 46 nude mice and randomly divided into curcumin group,radiotherapy group and curcumin plus radiotherapy group.Tumor size and apoptosis were detected by daily observation and TUNEL staining respectively.Results Curcumin inhibited proliferation and apoptosis of CD133 + rectal cancer cells when combined with radiotherapy.It also significantly increased the growth inhibition of rectal tumor and promoted the apoptosis of rectal cancer in vivo.MTT assay showed that after 24 hours,compared with that of radiotherapy group(14.6％ ± 1.0％),curcumin plus radiotherapy group (18.7％ ± 1.7％) inhibited the growth of the tumor(P ＜ 0.01).Annexin V/PI showed that curcumin plus radiotherapy group (28.8％ ±3.7％) was significantly different from the radiotherapy group(13.1％ ± 1.4％) in cell apoptosis (P ＜0.01).In vivo,after 6 days,tumor volume (521 ± 79) mm3 in curcumin plus radiotherapy group was significantly lower than that of radiotherapy group(717 ± 134) mm3 (P ＜ 0.01) ; TUNEL staining results indicated that the RCST in curcumin plus radiotherapy group (26.1％ ± 3.3％) were higher than that in radiotherapy group (12.0％ ± 2.1％) (P ＜ 0.01).Conclusions Curcumin significantly enhances the radiosensitizing effect for CD133 + rectal cancer cells.
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Objective To establish a radioresistant human cell line from esophageal squamous car-cinoma cells and detect the marker expression of cancer stem cells (CSCs). Methods A radioresistant hu-man esophageal squamous carcinoma cell line KYSE-150R was established by fractionated irradiation. Mor-phological changes from KYSE-150 to KYSE-150R were observed by phase- contrast microscopy. Karyotype analysis was performed by G-banding. The radiosensitivity of these two cell lines was assessed by colony for-marion assays. The cell cycle distribution was assayed by flow cytometry. CSCs markers of β-catenin and In-tegrin-β_1 were measured by Western Blot. Results The population doubling time of KYSE-150 and KYSE-150R were (23.62±0.23) hrand (25.90±0.55) hr, respectively (t =6.62,P=0.00). The numbers of chromosomes in KYSE-150R cells were increased and chromosome aberrations were observed. The SF_2, D_0, D_q and N values of KYSE-150R were all higher than those of KYSE-150. The ratio of D_0 values was 1.21. After irradiation, the number of S-phase cells of KYSE-150 increased from 45.35%±4.03% to 55.09%± 1.70% (t = -3.86,P=0.02) and G2/M phase cells decreased from 9.91%±3.83% to 1.15%±0.32% (t = 3.95, P = 0.02). However, no apparent change of cell cycle distribution for KYSE- 150R was observed. The expression levels of CSCs markers, β-catenin and Integrin-β_1 in KYSE-150R were about 2 times of those in KYSE-150. Conclusions The new cell line KYSE-150R is more radioresistant than its parental cell line KYSE-150. The CSCs in KYSE-150R is more than those in KYSE-150, which may suggest that CSCs is re-lated with the radioresistance.
ABSTRACT
AIM: To investigate the effect of 5 - fluorouracil ( 5 - FU ) on the expression of the stem cell marker CD133 on colon cancer stem cells. METHODS:CD133 expression on several colon cancer cell lines was detected by flow cytometry. The CD133 positive cells from DLD1 cells were separated by the method of magnetic activated cell separation. Colony assay was used to measure self - renew ability and MTS assay was used to detect the sensitivity to 5 - FU after separation. After 5 - FU treatment, the change of CD133 mRNA level was measured by qPCR. RESULTS: CD133 expression on the surface of colon cacner cell lines DLD1, HT29, SW480, HCT116, Lovo, RKO was 30.20% , 82.00% , 0.34% , 91.80% , 85.30% , 0.28% respectively. DLD1 cells had two obvious populations according to CD133 expression. CD133 positive cells were separated from DLD1 cells, the positive purity was 87.21% ±5.33% and the negative purity was 84.30% ±4.65%. CD133 positive cells formed more colonies with limited dilution colony assay (46.33% ±4.44% vs 31.00% ±2.00% , P <0.05). CD133 positive cells were less sensitive to 5 - FU compared to CD133 negative cells(20% less, P <0.01). 5 - FU at concentration of 1 mg/L upregulated CD133 mRNA expression in both DLD1 and HT29 cells, the relative quantity was increased from 1 to 1.684 ±0.012(P <0.01 )and 30.702 ±0.280 to 49.379 ±0.460(P <0.01) in HT29 and DLD1, respectively. CONCLUSION: Compared to CD133 negative cells, CD133 positive cells show more ability to form colonies in vitro, and are less sensitive to 5 - FU. 5 - FU upregulats the mRNA expression of CD133, resulting in the CD133 colon cancer stem cells enrichment during 5 - FU treatment.