| Carcinoma of the tongue, one of the most common oral malignant tumors, accounts for 40% of oral cancer. Over the past decade, the incidence of tongue cancer in China showed an upward trend year by year. Although the levels of diagnosis and the treatment on tongue carcinoma have being evidently improved in recent years, the survival rate of 5 years for patients with tongue carcinoma is still very low, which the main cause of poor curative effect and higher mortality is early invasion and metastasis. The tongue is an active muscle organ with abundant blood supply and lymph drainage, which facilitates its infiltrative growth and lymphatic metastasis at its early stage and thus makes it more difficult in its treatment. Recently, with the progress of the molecular biology, above all the advances in molecular targeted therapy, the treatment of oral cancer is becoming increasingly turned to chemical drugs targeted therapy. So, discussing the factors of tongue cancer invasion, to find effective, specific chemicals and to explore its effect, not only conducive to the chemical prevention of tongue cancer metastasis, but also to provide a new way to clinical treatment.In recent years, epidemiological investigation revealed that patients with a long-term administration of non-steroidal anti-inflammatory drugs (NSAIDs) had a significantly lower incidence of colorectal cancer, and reduced the rate of colorectal cancer metastasis and postoperative recurrence rate. The effect of NSAIDs that inhibit tumor mainly related to inhibition of the activity of cyclooxygenase-2 (COX-2). Studies have shown, COX-2 was strongly positive expression in eolorectal cancer, breast cancer, gastric cancer and other tumor tissues, especially in adenocarcinoma and squamous cell carcinoma which poorly differentiated and associated with local infiltration or distant metastasis. The COX-2 expression of different tumor cells derived from the same tissue types is different basing on their ability of invasion and metastasis to vary. Some studies shown that, COX-2 over-expression in colorectal cancer cells can promote invasion and metastasis, silencing COX-2 can reduce the invasion and metastasis of colorectal cancer cell. The molecular mechanism of tumor cell invasion and metastasis COX-2-induced are inseparable with the adhesion molecules, membrane folds of tumor cell, lamellipodia formation and extracellular matrix. Recent studies found that selective COX-2 inhibitor Celecoxib can inhibit tumor cell invasion by inhibiting the expression and activity of matrix metalloproteinase (MMPs), and become one of the hot studies that invasion and metastasis of cancer prevention, and for the relationship between celecoxib and the invasion and metastasis of tongue squamous cell carcinoma has not been reported in the other literatures.This study focuses on the inhibition of celecoxib on invasion of human tongue squamous cell carcinoma (HTSCC) Tca8113 cell, and initially study the mechanism of inhibition, with the purpose of revealing the invasion effect of celecoxib on the tongue squamous cell carcinoma to guide the use of drugs in clinic.Part I Study on the inhibitory effect of COX-2 inhibitor celecoxib on invasiveness of human tongue squamous cell carcinoma Tca8113 cellTaking Tca8113 cells grow well, inoculating on 6-well plate and conventional culture, waiting for covered with about 70% cells, we do the cell scratch, and then were cultured with 0,10μmol/L celecoxib for 24 h. then we can observe the different invasion capacity of human tongue squamous carcinoma Tca8113 cell accordance with the cells that migrate to the damaged area; In cell-matrix adhesion experiments, we detect the effect of adhesion ability between Tca8113 cells and extracellular matrix treatment with celecoxib using MTT method, By transwell chamber experiments, we detect the change of migration ability by counting the number of cells treated with different concentrations celecoxib for 24 h in vitro; Matrigel gel (Matrigel is extracted from Engelhreth-Holm-Swarm mouse sarcoma, and is rich in extracellular matrix protein, whose major components including laminin,Ⅳcollagen and proteoglycan sulfate and other substances) was tiled on the Transwell invasion chamber, using artificial recombinant basement membrane to simulate the invasion of tumor cells in vitro, to detect the effect of invasion which Tca8113 cells treated with different concentrations celecoxib for 24 h. Results:Scratch test showed that:the invasion of Tca8113 cells was strong, and the invasion could be inhibited by celecoxib; Cell-matrix adhesion test showed that celecoxib could against the adhesion between Tca8113 cells and extracellular matrix; The ability Tca8113 cells adhesion to Matrigel treatment with 10,20μmol/L celecoxib for 24 h decreased significantly (P<0.01), while the difference of 5μmol/L celecoxib compared with the control group was not statistically significant.The cell migration test show that, Tca8113 cells was incubated in the Boyden chemotaxis chamber for 24 h, and the number of migration cell was compared with the control group, the difference of 5μmol/L celecoxib group was not obvious (P=0.052); while 10,20μmol/L concentration group, the number of migration cell reduced significantly, and the difference was statistically significant compared with the control group (P=0.000); Artificial reconstituted basement membrane results show that:the number of Tca8113 cells treatment group through the polycarbonate membrane decreased significantly compared with the control group. After the treatment of 5,10,20μmol/L celecoxib, the number of Tca8113 cells through artificial basement membrane were 132.7±6.0 (P=0.073),68.3±6.5 (P=0.000),37.7±4.5 (P=0.000) respectively. So, celecoxib can inhibit the invasion of Tca8113, the inhibitory effect is in a dose-dependent, the maximal inhibition rate is 75.6%.Part II Celecoxib inhibit invasiveness of human tongue squamous Tca8113 cells through down regulating expression of MMP-2 and MMP-9Take growth well Tca8113 cells culturing normally for 24 h, add the culture medium containing different concentrations which the final concentrations is 0,5,10, 20,40μmol/L, treat for 24,48 h, then detect OD value using MTT to detect the inhibition of Tca8113 cell proliferation by celecoxib; With different concentrations (0, 5,10,20μmol/L) celecoxib treatment Tca8113 cells for 24 h, then extract the RNA of Tca8113 cell, reverse transcription and amplify, separated by agarose gel electrophoresis, to observe the effects of different concentrations of celecoxib on the expression of MMP-2, MMP-9mRNA in Tca8113 cells; Tca8113 cells were cultured in the culture medium containing 0,5,10,20μmol/L celecoxib for 24 h, taking the cell culture supernatants, detecting the activity of MMP-2, MMP-9 in Tca8113 cells with different concentrations of celecoxib culturing by gelatin zymography. Result: The MTT assay results showed that the inhibition of celecoxib on Tca8113 cell enhanced with its concentration and incubation time, the inhibition rate of 40μmol/L celecoxib on cell growth up to 82.2% after 24 h; while the inhibition rate of 20,40μmol/L celecoxib on cell growth up to 71.7% and 91.6% after 48h. RT-PCR electrophoresis results showed that, the expression of MMP-2 was decreased significantly by 10,20μmol/L celecoxib treatment (P=0.000),5μmol/L group is not different significantly compared with the control group (P=0.295). The expression of MMP-9 was decreased significantly too (P=0.007,5μmol/L group; P=0.000, 10μmol/L group; P=0.000,20μmol/L group). Gelatin zymography results showed that the activity of MMP-2, MMP-9 were inhibited treatment with 10,20μmol/L celecoxib compared with the control group, and was enhanced with increasing concentration, there is significant difference (P=0.000). While the activity of MMP-2 inhibition was not obvious (P=0.054), the activity of MMP-9 significantly inhibited (P=0.03) after the treatment of 5μmol/L celecoxib.Conclusion1. COX-2 inhibitor celecoxib can inhibite the invasion of human tongue squamous carcinoma Tca8113 cells significantly;2. The inhibition of celecoxib to human tongue squamous carcinoma Tca8113cell may be accomplish by inhibiting the activity of MMP-2 and MMP-9.
|
PDF Full Text Request