The Effect And Mechanism Of Lipopolysaccharide On Stemness Maintenance Of Liver Cancer Stem Cells

Objective Recent studies have shown that a subpopulation of cancer cells, termed cancer stem cells(CSCs) or tumor initiating cells(TICs) with characterizations of self-renewal,plasticity, dormancy, and metastasis, are responsible for tumor relapse after therapy.Due to the existence of liver cancer stem cell(CSCs), human hepatocellular carcinoma(HCC) can survive from many commonly employed cancer therapies. Hence, CSCs are considered to be a pivotal target for eradication of HCC. However, the mechanisms of maintenance and enhancement of CSC stemness remain unclear. In addition, although the importance of lipopolysaccharide(LPS) in promotion of HCC is well known, its role in the stemness maintenance of liver cancer stem cells is still unknown. Therefore,we study on the effect and mechanism of LPS on stemness maintenance of liver cancer stem cells(LCSCs).Methods1. Magnetic-activated cell sorting was performed to isolate CD133+ cells from human HCC cell lines. And the sorted CD133+ cells were cultured with PBS or LPS(10?g/m L)for 4 weeks. Then, expression of CD133 by the cells was measured by flow cytometry at time points of week 0, 1, 2, and 4.2. Expression of CD133 by the sorted CD133+ cells was measured by RT-PCR and Western blotting at time points of week 0, 1, 2, and 4.3. Clonogenic ability of the PBS or LPS-stimulated CD133+ cells was examined at time points of week 1, 2, and 4 using clonogenic assay, and tumorigenicity of CD133+cells was detected after cultured with PBS or LPS for a week. What's more, cell migration and invasion ability of the sorted CD133+cells was determined using the real-time cell analysis and transwell assay, respectively. And the chemoresistant ability of the PBS or LPS-stimulated CD133+ cells was also examined at time points of week1, 2, and 4.4. RT-PCR and western blotting was performed to detect the expression of HIF-1? at the end of week 1, 2 and 4 of culture. Then, the expression of HIF-1? and CD133 was detected after using HIF-1?-specific si RNA by RT-PCR and western blotting at the end of week 1 culture. 5. The activation of NF-?B in LPS-stimulated CD133+ cells was detected by western blotting analysis. RT-PCR and western blotting were applied to determine the expression of HIF-1? and CD133 after adding NF-?B inhibitor, BAY11-7082.6. Cell migration and invasion abilities of the sorted CD133+ cells, which were stimulated with PBS or LPS for 4 weeks cells, were determined respectively using the real-time cell analysis and transwell assay after adding HIF-1? small interfering RNA.Results1. Purity of the sorted CD133+ cells was 97.0%. A decrease in the ratio of CD133+ cells in a time-dependent manner was detected using flow cytometry. While, exposure to LPS resulted in a delay of reduction in the ratio of CD133+cells.2. RT-PCR and western blotting analysis also showed a decrease in CD133 expression by cultured cells in a time-dependent manner. Again, this decrease in CD133 expression was attenuated in cells cultured with LPS.3. There were significantly more clone numbers in LPS-stimulated group in comparison to the control group at each time point. And CD133+ cells displayed significantly stronger activity of tumorigenicity in comparison to those without LPS stimulation. What's more, LPS played an important role in maintaining the activities of migration and invasion of CD133+ cells, as well as their chemo-resistance.4. Exposure to LPS attenuated the reduction of HIF-1? by CD133+ cells at each time points of determination during the culture. And expression of CD133 was reduced by interfering HIF-1? expression.5. Blocking NF-?B activation with BAY11-7082(a NF-?B inhibitor) significantly reduced expression of HIF-1? and CD133 at both m RNA and protein levels in LPS-stimulated CD133+cells.6. Inhibition of HIF-1? expression with small interfering RNA decreased both migration and invasion activities of LPS-stimulated CD133+ cells.Conculsion Our results indicate that LPS, an important mediator in liver tumor microenvironment,contributes to the stemness maintenance of CD133+ liver cancer stem cells through NF-?B/HIF-1? pathway, suggesting that LPS represents therapeutic targets for making liver CSCs more sensitive to the anti-cancer treatments.