| Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide, and the third leading cause of cancer-related mortality. Surgical resection remains the most effective and practical treatment for HCC patients. Nevertheless, long-term survival remains unsatisfactory owing to the high rates of recurrence and metastasis.The cancer stem cells (CSCs) hypothesis stipulates that primary tumors are initiated and maintained by a small population of cancer cells with stem cell-like properties, such as self renewal, unlimited proliferation, and differentiation. Evidence suggests that in many malignances, including HCC, low-abundance CSCs are responsible for tumor relapse and metastasis. Since CSCs are involved in resistance and relapse to anticancer treatments, a selective targeting and eradication of CSCs is one of the most therapeutically important challenges.Cyclooxygenase-2 (COX-2) is not expressed constitutively, but induced by various extracellular or intracellular physiological stimuli. The up-regulation and over-expression of Cox-2 is mainly associated with inflammation, loss of apoptosis, unlimited proliferation, enhanced chemo-resistance, metastasis, and formation of new blood vessels finally leading to cancer. Previously, evidence suggests that COX-2 also plays a pivotal in CSCs biology. So pharmaceutical strategies directed towards COX-2 may help to reduce relapse and metastasis. Clinical trials further showed that chemo-preventive effect of COX-2 inhibitors in several malignances, such as colon cancer, lung cancer, breast cancer, and ovarian cancer.In present study, we explored the expression and biological function of COX-2 in liver cancer stem cells (LCSCs), and we sought to investigate whether COX-2 participate in the regulation of LCSCs and determine the specific effects of the selective COX-2 inhibitor celecoxib on LCSCs.Part I Biological characteristics of side population cells sorted from human hepatocellular carcinoma cell line HCCLM3Objective:To isolate side population (SP) cells from human hepatocellular carcinoma (HCC) cell line HCCLM3 with highly metastatic potential, and identify the biological characteristics.Methods:Four human HCC cell lines (HCCLM3, MHCC97-H, MHCC97-L, Huh7) with different metastatic potentials were cultivated by routine method. Single-cell suspensions were incubated with different concentrations of Hoechst 33342 alone or in the presence of 50 μM verapamil. Flow cytometry (FCM) was used to analyze the relative proportions of SP cells in each suspension. The SP gate was defined as the region where cells disappeared when cells were exposed to verapamil, which blocks Hoechst 33342 efflux. Hoechst33342 fluorescence intensity in SP cells and MP cells from HCCLM3 were detected by fluorescence microscope. Then SP and MP cell populations were compared in assays of self-renewal, proliferation, clonogenicity, migration, and invasion, and chemo-resistance in vitro, as well as in tumorigenicity assays in vivo. The two cell populations were compared in their expression of the cancer stem cell-associated genes Nanog, Oct4, Sox2, Klf4, and Sall4 using real-time quantitative PCR, and their expression of cancer stem cell markers ABCG2, CD133, CD90, EpCAM, CD44, CD13 using FCM.Results:Relative abundance of SP cells correlated directly with the metastatic potential of the HCC cell line:HCCLM3,16.3±2.2%; MHCC97-H, 8.4±0.7%; MHCC97-L,4.7±0.5%; and Huh7,1.0±0.3% (P< 0.05). Since HCCLM3 contained the highest proportion of SP cells further experiments were performed using these cells. Hoechst33342 fluorescence intensity in SP cells was significantly weaker than that of MP cells. SP cells isolated from HCCLM3 cultures showed significantly higher self-renewal, proliferation, clonogenicity and chemo-resistance than the corresponding MP cells, as well as higher migration and invasion ability in vitro and greater tumorigenicity in mice. Expression of all cancer stem cell-associated genes tested, except Sall4 and Oct4, was significantly higher in SP cells. And expression of all cancer stem cell markers tested by flow cytometry, except CD133, EpCAM and CD44 was significantly higher in SP cells.Conclusion:The proportion of SP cells correlates with metastatic potential. Fluorescence Activated Cell Sorting (FACS) is a feasible method to isolate SP and MP cells from HCCLM3cell line. And SP cells sorted from HCCLM3 show the characteristics expected of cancer stem cells, indicating that they may richen in liver cancer stem cells.Part Ⅱ Roles of COX-2 up-expression on cancer stem cell-like characteristics of SP cells in hepatocellular carcinomaObjective:To explore gene and protein expression of cyclooxygenase-2 (COX-2) in SP and MP cells from HCCLM3 cell line, and define the roles of up-expression COX-2 on cancer stem cell-like characteristics of SP cells in HCC.Methods:SP and MP cells were sorted from HCCLM3 by FCM. The expression of COX-2 was detected by real-time quantitative PCR and western blotting. LV-COX-2 and LV-NC were transfected into HCCLM3. LV-COX/SP and LV-NC/SP cells were sorted by FCM, and then analyzed by proliferation, and clonogenicity in vitro, as well as tumorigenicity assays in vivo. The expressions of COX-2, PTEN, and PDCD4 in LV-COX-2/SP cells were detected by real-time quantitative PCR and western blotting. The amount of PGE2 released into the medium was measured by using a special enzyme immunoassay kit.Results:The expression level of COX-2 mRNA in SP cells was 17.45 times of that in MP cells, and expression level of COX-2 protein was 9.23 times of that in MP cells. The proportion of SP cells was significant higher in LV-COX-2/HCCLM3,19.9±1.4% than that in LV-NC/HCCLM3,17.3±0.6%, and HCCLM3,16.9±1.8% (P<0.05). LV-COX-2/SP cells showed significantly higher proliferation and clonogenicity than LV-NC/SP and SP cells, as well as greater tumorigenicity in nude mice. The higher expression of COX-2 was observed in LV-COX-2/SP cells than LV-NC/SP and SP cells, while the lower expression of PTEN, and PDCD4, respectively, was observed in LV-COX-2/SP cells than LV-NC/SP and SP cells. The concentration of PGE2 released into medium was increased in LV-COX-2/SP cells in comparison with LV-NC/SP and SP cells (P<0.05).Conclusion:Up-expression of COX-2 enhances cancer stem cell-like characteristics of LCSCs by down-regulation of PTEN and PDCD4.Part III Effects of selective COX-2 inhibitor, celecoxib, on cancer stem cell-like characteristics of SP cells in hepatocellular carcinomaObjective:To explore the effects of celecoxib on cancer stem cell-like characteristics of SP cells in HCC.Methods:SP cells were sorted from HCCLM3 using FCM. The in vitro inhibitory effects of celecoxib at different concentrations and for different treatment time lengths on SP cells were observed with CCK-8 assay, the function of celecoxib on clonogenicity inhibition was observed with colony formation assay. The effect of celecoxib on apoptosis was studied by FCM. The in vivo tumor inhibition effect of celecoxib was evaluated in nude mice inplanted with SP cells. The expression of COX-2, PTEN, and PDCD4 were detected by real-time quantitative PCR and western blotting. The amount of PGE2 released into the medium was measured by using a special enzyme immunoassay kit.Results:Compared with control group, the absorption value decreased significantly with the increase of celecoxib concentration and exposure time. Celecoxib significantly inhibited the clonogenicity of SP cells, and induced cell apoptosis. Celecoxib showed significant inhibitory effect on the growth of transplanted tumor. The lower expression of COX-2 was observed in SP cells treated with celecoxib than untreated SP cells. And the higher expression of PTEN, and PDCD4, respectively, was observed in SP cells treated with celecoxib than untreated SP cells. The concentration of PGE2 released into medium was decreased in SP cells treated with celecoxib in comparison with untreated SP cells.Conclusion:Celecoxib suppresses cancer stem cell-like characteristics of LCSCs by up-regulation of PTEN and PDCD4. |
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