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@# Objective: To explore the resistance of CD133+ cells in HepG2 cell line to doxorubicin (DOX) and its mechanism. Methods: CD133+ cells were sorted by magnetic beads and CD133+ positive rate was detected by flow cytometry. MTT assay was used to detect the resistance to DOX-induced apoptosis of CD133+ cells. The expression of BCRP transporter mRNA was detected by RT-PCR. The expression of apoptosis-related proteins was detected by Western blotting. Immunofluorescence assay was used to detect the activation and transportation of P65 after DOX treatment. Results: Magnetic beads sorting could efficiently sort the CD133+ cells from HepG2 cells. MTT proliferation assay showed that CD133+ cells had stronger resistance to DOX than CD133- cells (P<0.05). Immunofluorescence showed that the activation rate and content of P65 in CD133+ cells were significantly higher than those in CD133- cells and HepG2 cells (P<0.05). The results of RT-PCR showed that the mRNA content of BCRP in CD133+ cells was significantly increased compared with CD133- cells and HepG2 cells (all P<0.05). Compared with HepG2 and CD133- groups, the expression of Bax and p53 in CD133+ cells was significantly decreased (P<0.05), while the expression of Bcl-2 and Survivin protein in CD133+ cells was significantly increased (P<0.05 or P<0.01). Conclusion: The molecular mechanism of high DOX-resistance of the CD133+ cell subsets in HepG2 cells is the high expression of the survival-related proteins NF-κB, Bcl-2, Survivin and the drug-resistance transporter BCRP, and low expression of apoptosis-promoting proteins p53 and Bax.
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The objective is to develop an easier technique for regenerating corpora cavernosa tissue through transplantation of human bone marrow-derived CD133+ cells into a rat corpora cavernosa defect model. We excised 2 mm × 2 mm squares of the right corpora cavernosa of twenty-three 8-week-old male nude rats. Alginate gel sponge sheets supplemented with 1 × 104 CD133+ cells were then placed over the excised area of nine rats. Functional and histological evaluations were carried out 8 weeks later. The mean intracavernous pressure/mean arterial pressure ratio for the nine rats (0.34258 ± 0.0831) was significantly higher than that for eight rats with only the excision (0.0580 ± 0.0831, P = 0.0238) and similar to that for five rats for which the penis was exposed, and there was no excision (0.37228 ± 0.1051, P = 0.8266). Immunohistochemical analysis revealed that the nine fully treated rats had venous sinus-like structures and quantitative reverse transcription polymerase chain reaction analysis of extracts from their alginate gel sponge sheets revealed that the amounts of mRNA encoding the nerve growth factor (NGF), and vascular endothelial growth factor (VEGF) were significantly higher than those for rats treated with alginate gel sheets without cell supplementation (NGF: P = 0.0309; VEGF: P < 0.0001). These findings show that transplantation of CD133+ cells accelerates functional and histological recovery in the corpora cavernosa defect model.
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AIM: To investigate the synergistic effect of resveratrol and 5-fluorouracil on osteosarcoma CD133+ cell subset.METHODS: Human osteosarcoma cell line MG-63 CD133+ cell subset and the corresponding CD133-cell subset were treated with resveratrol and 5-fluorouracil.After treatment, the viability of MG-63 cells was measured by MTT assay.The apoptosis of MG-63 cells was analyzed by flow cytometry.Activation of caspase-9 and caspase-3, the expression of Apaf-1, and the release of cytochrome C were evaluated by Western blot.The interaction between Apaf-1 and pro-caspase-9 was detected by co-immunoprecipitation.RESULTS: The cell death and apoptosis of MG-63 CD133+ cell subset induced by 5-fluorouracil were significantly weaker than those in the corresponding MG-63 CD133-cell subset.However, co-treatment with resveratrol significantly enhanced the effect of 5-fluorouracil on inhibiting the viability of MG-63 CD133+ cell subset.Mechanically, treatment with resveratrol upregulated the expression of Apaf-1.Transfection with Apaf-1 siRNA abolished the synergistic effect of resveratrol and 5-fluorouracil in MG-63 CD133+ cell subset.In addition, the results of co-immunoprecipitation indicated that the combination of resveratrol and 5-fluorouracil significantly induced the formation of Apaf-1/pro-caspase-9 complex, leading to the activation of caspase-9 in MG-63 CD133+ cell subset.CONCLUSION: Resveratrol enhances 5-fluorouracil-induced apoptosis of osteosarcoma CD133+ cell subset by promoting the formation of Apaf-1/caspase-9 complex.
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AIM:To investigate the role of imperatorin in reversing the resistance of the PC 9 CD133+cell sub-sets to gefitinib.METHODS:MTT assay was performed to evaluate the viability of PC 9 cells treated with imperatorin and gefitinib.The expression of c-met, activation of caspases and phosphorylation of epidermal growth factor receptor (EGFR), PI3K and AKT in the PC9 cells treated with imperatorin and gefitinib were determined by Western blot .The percentage of CD133 +cell subsets population and the apoptotic rate of the PC 9 cells treated with imperatorin and gefitinib were analyzed by flow cytometry .RESULTS:The sensitivity of the PC 9 CD133 +cell subsets to gefitinib was significantly lower than that of the PC9 CD133 -cell subsets.Treatment with gefitinib alone significantly inhibited the protein levels of EGFR /PI3K/AKT in the PC9 CD133 -cell subsets but not the PC 9 CD133 +cell subsets .Treatment with gefitinib alone increased the percentage of CD133 +cell subsets population in the PC9 cells.However, combination of gefitinib with imperatorin signifi-cantly inhibited the enrichment of CD 133 +cell subsets population .Imperatorin down-regulated c-met expression , sugges-ting the c-met was the target of imperatorin in the PC9 CD133 +cell subsets.The results of MTT assay, Western blot analy-sis and flow cytometry indicated that imperatorin increased the gefitinib induced inhibition of PI 3K/AKT protein levels by down-regulating the expression of c-met, which subsequently induced the cleavage of caspases and apoptosis in the PC 9 CD133 +cell subsets.CONCLUSION:Imperatorin increases the sensitivity of lung cancer CD 133 +cell subsets to gefitinib by down-regulating the expression of c-met, and the synergistic anti-tumor effect exists between imperatorin and gefitinib .
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Background and purpose:The most serious aspect of cancer is the emergence of metastases in organs distant from the primary tumor, since most deaths from cancer are due to metastases. In recent years, a series of studies has been undertaken both in vitro and in vivo, and demonstrated that the CD133+ hematopoietic stem cell is effective in increasing mice tumor cell metastases, but the efficacy of CD133+ stem cells is not well studied for human cancer.We investigated the effects of CD133+ stem cells on the proliferation, adhesion and migration in human colorectal neoplasm cell line SW480.Methods:CD133+ cells were separated from fresh cord blood mononuclear cells(MNC) by Ficoll density gradient centrifugation and magnetic activated cell-sorting system(MACS).The treatment group was the co-culture of CD133+ cells and SW480, the control group used the same culture medium without CD133+ cells. The effect of CD133+ cells on proliferation in SW480 cell was measured by MTT assay, the migration abilities of SW480 cell were assayed in Transwell cell culture, Cell adhesion assay was carried out in a 96 microplate well precoated with fibronection.Results:CD133+ cells could significantly improve the growth ability of SW480 cell, not only the ability of proliferation, but also the morphology. The morphology of the cells was remarkably changed. Irregular nucleus, double nucleus and polymorphic nucleus appeared in the treatment group, and the cells were shaped as polygon-like and leptosomatic. In the cell adhesion assay, A 490 of the treatment group was 0.11?0.01, A 490 of the control group was 0.05?0.01(P﹤0.05).Conclusions:Our results indicate that the CD133+ cell is effective in increasing tumor cell proliferation and invasion. Hematopoietic stem cell may play an important role in the invasion and metastasis of colorectal neoplasm.