Study On The Mechanism Of Notch Signaling Pathway In Curcumin - Mediated Photodynamic Therapy For Cervical

Cervical cancer,second only to breast cancer,has become the second big malignant tumor endanger the lives of women for its high incidence and the death toll. It s the one of the most important topic in the field of medicine. This experiment aim at find the effect on cervical cancer transplated tumor in vitro by using photodynamic therapy(PDT) combined with photosensitive traditional Chinese medicine-curcumin. And provide reference for clinical treatment by explore its mechanism.Objective:1.Develop cervical cancer Me-180 cells in vitro,construction the cervical cancer transplantation tumor model.2. Validate the effect of curcumin mediated PDT to the tumor volume of cervical cancer transplanted tumor in vitro.3. Clarify the mechanism of the curumin-PDT in the treatment of cervical cancer。Method:Inject 100μI. cervical cancer cell suspension to the back of each BALB/C mude mice. After 2-3 days incubation period, we can see a pea-sized transplantation tumor appeared in vaccination. Assumes the circular or elliptic.and this means the successful establishment of cervical cancer transplantation tumor model in vitro. When the tumor diameter of about 0.8 to 1.0cm, subsequent experiment was carried out. Experiment was divided into 5 groups, model group, Pure light group, the curcumin group, curcumin-PDT group as well as the blocker group. Each group of 12, and a total of 60. Model group:100 μL normal saline was injected around the tumor and don’t do any processing. Single light group:100 μL normal saline was injected around the tumor and give the single light which the wavelength is 425nm. The end of the optical fiber to the surface of tumor is about 2-3cm,Exposure area is about lcm2 and irradiation time is 20min. Made the light exposure dose to be 100J/cm2. Curcumin group:100μL curcumin solution which the concentration is 5OμM/L was injected around the tumor and give natural light. Curcumin-PDT group:100μL curcumin solution which the concentration is 50 u M/L was injected around the tumor and treated with PDT 6 hours later. Blockers group:According to the measurement of 100mg/Kg,Intraperitoneal injection of DAPT. And equal amounts of curcumin solution was injected around the tumor and treated with PDT 6 hours later. The day after treatment,6 nude mice was sacrificed and record the tumor size of the other 6 mice in each groups. Calculate tumor volume of every mice in each groups at different time. Oberve the change of tumor size and record it. The pathological change of each group were observed by HE staining, immunohistochemistry and Westen-blot was used to detect the expression of Notch-1、NF-κB and VEGF.Result:1. Injection of cervical cancer Me-180 cell 1 to 2 days after suspension,There is a goitre, grain size, gradually increasing with time, Prompt the model building success.2. The effect curcumin-PDT on cervical cancer transplanted tumors in vitro, same time comparison between each groups:model group showed a trend of increase tumor volume after treatment。compared with model group, The tumor volume of curcumin group and curcumin-PDT group were statistical difference in the frist day (P<0.05);In the third day,each group were statistical difference compare with model group (P<0.05), and the curcumin group and curcumin-PDT group were significant difference (P<0.01). In the seventh day, each group were statistical difference compare with model group (P<0.05), and the curcumin group and curcumin-PDT group were significant difference (P<0.01). In the 14th day, curcumin group statistical difference (P<0.05).while curcumin-PDT group was significant difference compare with model group (P< 0.01). In the 21th day, curcumin group statistical difference (P<0.05).while curcumin-PDT group was significant difference compare with model group (P<0.01). compare within the group at different time:Compared with the tumor volume before treated, model group have statistical difference after 14 days(P<0.05), and a significant difference was found in 21 days(P<0.01). Pure light group:statistical difference was found in 14 days(P<0.05), and a significant difference was found in 21 days(P<0.01). curcumin group:statistical difference was found in 14 days(P<0.05), and a significant difference was found in 21 days (P<0.01). Curcumin-PDT group:a significant difference was found in 21 days (P<0.01).3. HE staining results:Tumor cells in model group showed nest arrangement. cell composition is relatively single, Nucleoplasm ratio imbalance, Cell boundary is not clear,the size of nuclear are not the same size, assumes the circular or ovoid, chromatin is rough nuclear fission as common and occasional necrosis. Compared with model group,the cell in the each treatment groups arranged relatively sparse, shrinkage, nucleoplasm ratio decrease, part of the nucleus pycnosis, vesicular nuclei and nuclear week empty dizzy can be seen. Mitosis are rarely compare with model group and PDT-curcumin group are obviously.4. immunohistochemical results:model group Notch-1 expression is higher compared with model group, Notch-1 expression in each groups are decreased, with statistical significance (P<0.01). the compared with model group, NF-κB expression in the rest of the two groups were increasing and have statistical difference (P<0.01). Compared with curcumin-PDT group, the expression of NF-κB in blocker group are declined significantly (P< 0.05). The expression of VEGF in model group is low,the expression in the rest of the four treatment group were higher, and have statistically difference (P< 0.01), the most significant can be observed in the curcumin-PDT group (P< 0.01). The expression of VEGF in the blocker group was decrease compare with the curcumin-PDT group (P<0.01).5.Westen blot results: compared with model group, Notch—1 in Curcumin-PDTgroup was significantly reduced (P < 0. 01). Compared with curcumin - PDT group,Notch-1 expression in blocker group was increased, with statistical difference(P < 0. 01). Compared with model group, the expression of NF-kB in curcumin - PDTgroup are decreased, NF-kB expression in blocker group is increase compare withcurcumin - PDT group, (P<0. 05). Compared with model group, The expression of VEGFin Curcumin-PDT group are significantly rise compared with model group (P < 0. 01)The expression in blocker group is rise also (P < 0. 05). Compared with curcumin-PDT group,VEGF expression in blocker group is reduced,and have statisticallysignificant difference (P < 0.05).Conclusions:1.photodynamic therapy has the inhibitory effect for cervical cancer transplanted tumor in vitro.2. Notch-1 expression in treatment group are reduced compared with model group, and this indicated that inhibition of Notch-1 expression may has inhibitory effect on tumor growth.3. Notch-1/NF-κB/VEGF signaling pathway may plays an important role in cervical cancer.