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The Experimental Study of the Effect of Periplocin on the Proliferation of MDA-MB-231 in Vitro and in Vivo

Author: ZhangJianBin
Tutor: DanBaoEn；AiJun
School: Hebei Medical University
Course: Clinical Laboratory Science
Keywords: MDA-MB-231 Cortex Periplocae Periplocin stat3 IL-6 IL-8 Nude mice
CLC: R285.5
Type: Master's thesis
Year: 2011
Downloads: 33
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Abstract

Objective: To study the Cortex Periplocae extract Periplogenin (periplocin extracted from cortex periplocae, CPP) on human breast cancer cells MDA-MB-231 apoptosis and invasion of human as well as in vivo anti-tumor effect, analysis Periplogenin stat3 expression in MDA-MB-231 cells, the impact and stat3 gene expression regulation, anti-tumor mechanism of action studies periplocin, as a clinical anti-tumor drug development for the CPP to provide experimental basis. Method: an application with four methyl thiazolyl tetrazolium (MTT) method to detect different concentrations of CPP (0.625,1.25,2.5,5,10,20 μg / ml) for different times (24h, 48h and 72h) on MDA-MB -231 proliferative response inhibition. 2 light microscope to observe changes in cell morphology CPP treated; The Rui Ji staining CPP after changes in cell morphology. 3 immunocytochemistry assay the CPP (2.5μg/ml) after cell expression of p-Stat3; 4 cell scratch assay CPP (2.5μg/ml) cells before and after treatment invasion can change. 5 by reverse transcription - polymerase chain reaction (revers transcription PCR, RT-PCR) semi-quantitative detection of the CPP (2.5μg/ml) for different time (0h, 6h, 12h and 24h), MDA-MB-231 cells gene of stat3 changes in the expression of IL-8. 6 detected by ELISA CPP (2.5μg/ml) the role of MDA-MB-231 cells in IL-6 secretion changes. 7 CPP human breast cancer cells cells MDA-MB-231 tumor-bearing nude mice inhibitory effect on STAT3 signaling pathway. Washed with PBS solution, and MDA-MB-231 cell density was adjusted to 5 × 10 ~ 6/ml, subcutaneously in the right back of nude mice inoculated with MDA-MB-231 cells (0.2 ml containing 10 ~ 6), and the establishment of MDA -MB-231 cells, tumor-bearing nude mice were randomly divided into 2 groups of 10. Next to the experimental group tumor 0.2ml injection of CPP (30mg/kg), once a day, the control tissue to give 0.2ml PBS buffer peritumoral injection. Observed in nude mice and tumor growth, tumor size measured every 3 days. Administered for 12 days, eagerly mice were sacrificed, and the removal of the tumor tissue, respectively, said the tumor weight, tumor volume was measured inhibitory rate was calculated. RT-PCR to detect the experimental group and the expression on the stat3 the tumor tissue, tumor tissue p-stat3 expression detected by Western blot. Results: 1 CPP 0.625-20μg/ml concentration range can significantly inhibit human breast cancer MDA-MB-231 cells in vitro proliferative response, a concentration and time-dependent inhibition rate, increase with the concentration and duration of cell proliferation The inhibition rate rises, which of 20μg/ml Periplogenin 72 hours maximum inhibition rate was 70.25%. The different concentrations Periplogenin statistically significant differences in the rate of inhibition of MDA-MB-231 cells (p lt; 0.05). Morphogenesis 2 CPP role in MDA-MB-231 cells apoptosis preliminary change cell rounding, cytoplasmic concentrated loose chromatin, nuclear fragmentation, cytoplasmic staining lighter, and fusiform cells between the control cells, morphological irregular, the cytoplasm is deep blue, nuclei were larger, irregular, showing multiple nucleoli. Immunocytochemistry analysis showed that MDA-MB-231 cells p-Stat3 protein was localized in the nucleus, brownish-yellow granular. CPP after the decline in rate and the level of p-Stat3 expression (p lt; 0.05). 4 cells scratches experimental results show that the CPP role in cell invasion ability of MDA-MB-231 cells in vitro decreased. Semi-quantitative RT-PCR test results show that cell gene of stat3 With the extension of the CPP processing time, processing time group (0h, 6h, 12h and 24h), IL-8 mRNA expression levels decreased gradually with the untreated group (0h ) cells, the difference being statistically significant (p lt; 0.05). 6 ELISA analysis showed that, compared with the control group, CPP (2.5μg/ml) for 24 hours, MDA-MB-231 cells the cells secrete amount of IL-6 was 230.4 ± 3.4pg/ml, compared with the control group 5 12.2 ± 5.3pg/ml significantly reduced, and the difference was statistically significant (p lt; 0.05). 7 the establishment of human breast cancer cells MDA-MB-231 cells in nude mouse model, 100% of the rate of tumor formation. Compared with the control group, the experimental group, slow-growing tumors in nude mice. Tumor weight and volume of the experimental group was significantly lower than the experimental group, the average tumor weight of tumor tissue (p lt; 0.05) 1.437 ± 0.398g, tumor weight of the control group 3.458 ± 0.675g, the inhibition rate was 58.44%. RT-PCR results was stat3 expression level of tumor cells of the experimental group was significantly lower than the control group (p lt; 0.05). Western blot analysis showed that p-Stat3 protein expression in the tumor tissue of the experimental group than the control group. Conclusion: 1 CPP in a certain concentration range, could significantly inhibit the proliferation of breast cancer cells MDA-MB-231, a time-and dose-dependent manner. 2 CPP can induce human breast cancer cells MDA-MB-231 produce morphological changes of apoptosis; CPP inhibits MDA-MB-231 cells Stat3 signal transduction pathway, reducing the MDA-MB-231 cells tyrosine phosphorylation of Stat3 level. 4 CPP after periplocin can significantly inhibit the proliferation and invasion of MDA-MB-231 cells, and its mechanism may autocrine IL-6 and stat3 phosphorylation blocked stat3 signaling pathway by inhibiting cell IL-8 down expression. 5 CPP in human breast cancer cells MDA-MB-231 strong in vivo anti-tumor effect. CPP can reduce the protein expression of p-stat3 the xenograft tumor tissue. CPP in the body can significantly inhibit the proliferation of MDA-MB-231, its mechanism of inhibition of Stat3 signaling pathway.

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