Research Of Notch Inhibitors In The Treatment Of Tongue Cancer

Tongue cancer is one of the most common oral cancer, generally refers to malignant tumor which occurred in the front of 2/3 of the tongue and squamous cell carcinoma is more common. Tongue as an important part of human once occur lesions would seriously affect the patient’s life as well as facial appearance. Our previous studies have shown that Notch signaling pathway and its target genes as an oncogenic genes in salivary gland adenoid cystic carcinoma. Knockdown these genes expression could lead the cancer cells proliferation, migration and invasion ability decreased and increased apoptosis. There were a lot of studies to make use of Notch signaling pathway inhibitors especially gamma secretase inhibitors(GSI) to treat solid tumors, however,apply them in the research of oral cancer is relatively rare. On the basis of our previous studies, these research used immunohistochemical method proved that the Notch signaling pathway had a high expression in the tissues of tongue cancer. Used gamma secretase inhibitors(GSI)on the tongue cancer cells(CAL- 27), screened out effective inhibitors. Used them acted on tongue cancer cells to study the change of the cells proliferation and apoptosis in vitro and in vivo. To explore the change of downstream genes and its possible mechanism. This study could be divided into two parts:一、 To validated the expression of Notch signaling pathways in the tissue of tongue cancer, and screened out the effective Notch inhibitorsAim: To study the expression of Notch signaling pathway in tongue cancer tissue, and screen out effective Notch inhibitors.Methods: Collected the paraffin sample of tongue carcinoma tissues and adjacent tissues and did immunohistochemistry. Read and scored by two pathologists then statistics. Used semilog concentration diluted Notch inhibitors then acted on tongue cancer cell CAL- 27 after 72 h, utilized CCK 8 method to detected the influence of the cell vitality, and used Graphpad Prism5 software calculated the IC50. At the same time used the inverted microscope observed the cellular morphological changed, screened the effective inhibitors.Results: Notch1 and Notch2 in tongue cancer tissues expressed higher than adjacent tissue. CCK 8 method screened out two kinds of gamma secretase inhibitors which had obviously inhibiting effect to tongue cancer cell(CAL-27): the FLI-06 and JLK 6.Calculated IC50 of the inhibitors: FLI-06 about 2.90 μM and JLK 6 about 17.51 μM,and the treatment group of tongue cancer cells had morphologic change.Conclusion: The expression of the Notch signaling pathway in tongue cancer tissue were increased. Two kinds of effective gamma secretase inhibitors: FLI- 06 and JLK 6,effectively restrained the tongue cancer cell CAL- 27.二、 To observed the effect of Notch signaling pathway inhibitors on tongue cancer cells in vitro and vivo.Aim: To investigated the influence of cell function when Notch signaling pathway inhibitors acted on tongue cancer cells in vitro and in vivo.Methods: The first part we have been screened out effective inhibitors(FLI-06 and JLK6) in CAL-27, respectively used the CCK-8 method, plate clone formation experiments study the change of proliferation ability of cells. Flow cytometry studied the change of cell cycle and apoptosis. TUNEL method detected apoptosis in morphology. Real-time PCR and Western blot were used to detect related m RNA and protein change. Tongue cancer xenograft model were used to observe the treatment effect of FLI-06.Results: After gamma secretase inhibitors acted on the CAL- 27, used CCK8 method and plate clone experiments showed that the proliferation were restrained. And flow cytometry detection found that the late apoptosis rate and early apoptosis rate of the experimental group were higher than the negative control group, and observed the cell cycle blocked. TUNEL method showed that the apoptosis rate of treatment group was increased. Real-time PCR and Western blot method showed the expression of Notch receptors and their target genes of treatment group was decreased. The tumor volume of treatment group less than the control group.Conclusion: The two kinds of gamma secretase inhibitors(FLI- 06 and JLK 6)inhibited the proliferation of tongue cancer cells, blocked the cell cycle and cell apoptosis rate were increased and suppressed the Notch pathway related protein expression. Notch signaling pathway inhibitors(FLI-06) could inhibited tumor growth in vivo.