| Objective:1.To investigate the apoptosis-inducing effect of Solarnum Lyratum Thunb water extract (SLE) and its molecular mechanism on human colorectal cancer HT29 cells.2.To explore the impact of SLE to Notch 1 gene.Methods:1. To obtain water extract of Solanum Lyratum Thunb, the whole dried herbs of Solanum Lyratum Thunb were immersed, decocted, filtered and concentrated.2. Cultured human colorectal cancer HT29 cells in vitro which were randomly divided into the normal control group, SLE-treated groups (1.25,2.5,5mg/mL) and HCPT (10μg/mL) group (the positive control group).3. The cell proliferation inhibitory rate and IC50 values were evaluated by cytotoxic (MTT) assay after treated with SLE for 24h,48h and 72h.4. After treated human colorectal cancer HT29 cells with SLE for 24h, morphological changes of apoptosis were observed by using optical microscope and electron microscope.5. After treated human colorectal cancer HT29 cells with SLE for 24h, the cell apoptosis rate was detected by FCM Annexin V/PI dual-staining.6. After treated human colorectal cancer HT29 cells with SLE for 24h, the expressions of caspase-8 and Notch 1 mRNA were assessed by semi-quantitive RT-PCR.Results:1.SLE displayed strong proliferation inhibitory effect in a dose-and-time-dependent manner against human colorectal cancer HT29 cells. After treated with 1.25,2.5 and 5mg/mL SLE and HCPT(10μg/mL) for 24h, the inhibitory rate was (14.08±3.02)%, (35.98±3.62)%, (59.18±5.84)% and (52.90±5.05)% respectively. After treated for 48h, the inhibitory rate was (24.09±3.90)%, (57.15±3.99)%,(78.95±5.70)% and (71.61±5.32)% respectively. After treated for 72h, the inhibitory rate was (45.61±6.22)%, (70.05±2.46)%, (89.23±2.14)% and (87.83±2.01)% respectively. After treated with SLE for 24h,48h and 72h, the IC5o value was 3.83,2.29 and 1.42 mg/mL respectively.2.After treated with SLE and HCPT,we could see the cell proliferation slowed down, the cells shrinked, floated and didn't adhered to the wall under the optical microscope. In the electron microscope, the SLE induced cells shrinkage, chromatin condensation and margination, nuclear fragmentation, and apoptotic bodies. No changes were observed in control group.3. The effect on cell apoptosis:the apoptosis rate of control group on human colorectal cancer HT29 cells was (3.43±0.40)%,the SLE groups and HCPT group were (9.14±1.59)%, (21.55±2.03)%, (40.10±2.53)% and (39.67±2.82)% respectively,compared with control group,the apoptosis rate on human colorectal cancer HT29 cells increased obviously in SLE groups and HCPT group(P<0.01), and showed in a dose-dependent manner.4. The expression of caspase-8 mRNA was up-regulated,and the expression of Notch 1 mRNA was down-regulated in the human colorectal cancer HT29 cells of the SLE-treated groups. After treated with 1.25,2.5 and 5mg/mL SLE for 24h, the ratio of RT-PCR products of caspase-8 toβ-actin was 0.382±0.023,0.545±0.045 and 0.723±0.015 respectively. The activity of Caspase-8 mRNA in the SLE-treated groups was higher than that of the control group 0.204±0.043 significantly (P<0.001). The ratio of RT-PCR products of Notchl toβ-actin was 0.518±0.019,0.437±0.024 and 0.239±0.041 respectively. There was significant difference in SLE groups, compared with control group 0.648±0.019 (P<0.01 or P<0.001)Conclusion:1. SLE displays strong proliferation inhibitory effect in a dose-and-time-dependent manner against human colorectal cancer HT29 cells.2. SLE can induce apoptosis of HT29 cells and the apoptosis rate shows in a dose-dependent manner. Inducing-apoptosis may be one of mechanisms of SLE inhibiting the proliferation of HT29 cells.3. SLE can up-regulate the expression of caspase-8 mRNA. It indicates that the effect of SLE on inducing-apoptosis of HT29 cells may be related to the pathway of Death receptor.4. SLE can down-regulate the expression of Notchl mRNA, suggesting that the SLE may influence the Notch signaling pathway to inhibit Colorectal cancer cell proliferation and induce apoptosis. |
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