| Background and Objective:Gastric cancer is a major mortality cause in the world, and this disease is often at an advanced stage at presentation, The 5-year survival rates are 70-75% for stage Ⅰ disease, and drops to 35% for stage Ⅱ. Much efforts were undertaken to improve therapies and survival. Chemotherapy is one of the primary treatments against gastric cancer. However, chemotherapy strategies are not optimal. Indeed, hypoxia is one of the characteristics of solid tumors, including gastric cancer, and this state induce chemotherapy resistance.5-fluorouracil (5-Fu) is an antimetabolite chemotherapy drug. It inhibits the thymidylate synthase, blocking the transformation of deoxy-uridine monophosphate into deoxy-thymidine acid, resulting in cell death through a decreased DNA synthesis and S-phase arrest. Clinical trials showed that regimens containing 5-Fu improve survival to gastric cancer, but that local failure and distant metastases still occur. In vitro, previous studies showed that hypoxia induced a resistance of cancer cells against 5-Fu.Recent studies showed that very small amounts of cancer stem cells were present in tumor tissues, and that these cells had an unlimited self-renewal ability and unlimited differentiation potential. It is increasingly believed that these cancer stem cells are the cause of metastases, tumor recurrence, and resistance to drugs and radiations. However, at present, very few is currently known about these cells, and their phenotypic marker profile has not been precisely defined yet, impairing the possibility to purify them, so we use some cancer stem cell markers which closely associated with stem cells to investigate.Celecoxib is a non-steroidal anti-inflammatory drug, and a selective cyclooxygenase (COX)-2 inhibitor with anti-inflammatory and analgesic effects. Previous studies showed that celecoxib could be used for cancer treatment, but the involved mechanisms are poorly understood.Therefore, the aim of the present study was to assess the effects of celecoxib on hypoxic gastric cancer SGC7901 cells in vitro and vivo and the possible mechanisms. Furthermore we aimed to assess if celecoxib could reduce the hypoxia-induced resistance against 5-Fu in these cells in vitro and vivo, as well as the possible mechanisms. Results from the present study might improve the treatment of gastric cancer and improve the survival of these patients.Methods:1.SGC7901 cells were inoculated in RPMI1640 medium and were subcultured regularly,The chemical hypoxia agent CoCl2 (150μmol/L) was used to simulate the hypoxic microenvironment of solid tumors. Cells in the logarithmic growth phase (200μL) were inoculated in 96-well culture plates at a cell density of 2×105/mL. SGC7901 cells were divided into four groups:hypoxic control group,5-Fu group, celecoxib group and 5-Fu/celecoxib combination group. After treatments, The proliferation inhibition rates of different concentrations of 5-Fu and celecoxib in gastric cancer cells under hypoxia were determined by MTT, Protein and mRNA expression of HIF-2α, ABCG2 and OCT-4 in each group were determined by immunohistochemistry, RT-PCR and western blot.2.The tumor-bearing nude mice model was established, the mice were divided into four groups:blank control group,5-Fu group, celecoxib group and combination groups. After treatments, Mice were killed by cervical dislocation, the tumor was stripped with scissors, the tumor weight was documented. The weight change and the tumor inhibition rate in each group were calculated. Immunocytochemistry, RT-PCR, Western-blot methods were used to observe HIF-2α, ABCG2, OCT-4Protein and mRNA expression in tumor mass of all groups.Results:l.The proliferation of hypoxic SGC7901 gastric cancer cells was obviously inhibited by different concentrations of 5-Fu and celecoxib in a dose-dependent manner. Cells were in the logarithmic growth phase within 48 hours after inoculation. The IC50 of 5-Fu was 200 mg/L, while the IC50 of celecoxib was 100 μmol/L. Growth inhibition rates were 52.61%,46.1% and 66.09% in the 5-Fu, celecoxib and combination groups under hypoxic conditions, the inhibition rate was higher in the combination group, there was statistically significant difference between the combination group and 5-Fu group and celecoxib group (F=35.237, P<0.05). HIF-2a, ABCG2 and OCT-4 expression (mRNA and protein) was gradually lower from the 5-Fu group (the highest), to the hypoxia control group, and to the celecoxib and combination groups (both the lowest) (mRNA:HIF-2a F=819.242, ABCG2 F=515.693, OCT-4 F=1929.264, P<0.01. Protein:HIF-2αF=4788.138, ABCG2 F=1739.794, OCT-4 F= 25656.397, P<0.01)2.5-Fu, celecoxib and the combination group could inhibit the growth of implanted gastric cancer, the mean tumor weight and volume of tumor mass of 5-Fu group was less than the control group, but the differences was not significantly(P>0). 05), the mean tumor weight and volume of tumor mass of celecoxib group and combination treatment group was significantly less than that in the control group (P<0.05), the mean tumor weight and volume of tumor mass in the combination treatment group was significantly less than that in the 5-Fu group and celecoxib group (P<0.05). The inhibition rates were 26.36%,59.70%,88.37%, combination group had the highest inhibition rate. When compared with the other groups, the difference between combination group and 5-Fu group, the difference between celecoxib group and 5-Fu group were statistically significant (P<0.05). HIF-2a, ABCG2, OCT-4 mRNA and protein expression levels were high in blank control group, HIF-2a, ABCG2, OCT-4 mRNA and protein expression levels further increased in 5-Fu group. But in the celecoxib group and the combination group, the expression showed lower levels, compared with the control group,5-Fu group, celecoxib group and the combination group showed the statistically significant differences (P<0.01).Conclusion:1. Results suggest that celecoxib has anti-tumor effects in vitro and vivo, inhibiting tumor growth through inhibiting HIF-2a, ABCG2 and OCT-4.2. The 5-Fu/celecoxib combination had a synergic effect on tumor growth inhibitionin vitro and vivo. The mechanism of this effect may be related to reduced chemotherapy resistance and enhanced chemotherapy effect through inhibition of HIF-2α, ABCG2 and OCT-4. |
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