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Inhibitory Role of Plasmodium Yoelii Circumsporozoite Protein on the Proliferation and Survial of Tumor Cell

Author: DingYan
Tutor: XuWenYue
School: Third Military Medical University
Course: Pathogen Biology
Keywords: Circumsporozoite protein Nuclear transcription factorκB cancer cell proliferation apoptosis
CLC: R730.5
Type: Master's thesis
Year: 2010
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Abstract


Cancer is mainly account for the death of patients in clinical. The traditional method of surgical resection and chemoradiotherapy achieved a certain effect, however, 5-year survival rate of the tumor is still low. Therefore, developing new cancer treatment come to badly in need. Continuous activation of NF-κB is a significant mechanism of tumor cell survival, proliferation and migration, and inhibition of NF-κB activity is an important strategy for cancer therapy. Evidence showed that the circumsprozoites protein（CSP） of malaria sporozoite inside the parasitophorous vacuole could transfer to the cytoplasm by using its pexel domain, and inhibit activation of NF-κB in liver cell and respiratory burst of Kupffer cells. In view of this, we hypothesized that CSP might inhibit tumor cell proliferation and survival by blocking the activation of NF-κB.In this study,we firstly amplified the coding sequence of CSP from the sporozoites of Plasmodium yoelii BY265, and cloned it into pFLAG-CMV8, then the constructed recombinant pFLAG-CMV8-CSP was transfected into human colon cancer cell line SW480 and human hepatocarcinoma cell line HepG2 with Lipofactamine2000TM to observe the influence of CSP on the survival and proliferation of two kinds of tumor cells. Then recombinant plasmid pFLAG-CMV8-CSPΔNLS lack of nuclear localization sequence（Nuclear localization signal, NLS） of CSP and recombinant plasmid pFLAG-CMV8-CSP NLS only containing CSP NLS were also constructed, and their effect on NF-κB activation of tumor cells was investigated using NF-κB reporter assay and ,EMSA（Electrophoretic mobility shift assay）.1 Construction and expression of recombinant plasmid pFLAG-CMV8-CSP in cancer cell:The full length of the coding sequence of CSP was reverse transcribed and amplified by RT-PCR with sporozoite total RNA as template,and a fragment of 1281bp in length was obtained , cloned into pFLAG-CMV8 , and the correct of insert was confirmed by sequencing. Indirect immunofluorenscence staining with rabbit anti-CSP showed CSP mainly distributed in the cytoplasm and membrane.2 CSP inhibit tumor cell proliferation and survival（1） CSP inhibit the proliferation of tumor cells:Compared to the control transfected with pFLAG-CMV8, the proliferation of SW480 and HepG2 transfected with pFLAG-CMV8-CSP were markedly reduced, and the effect exhibited a dose-dependent manner.（2）CSP induced the apoptosis of tumor cellsAnnexin V-FITC apoptosis assay showed that the apoptosis of HepG2 transfected with pFLAG-CMV8-CSP was significantly higher than of control transfected with the vector pFLAG-CMV8（p <0.01）, indicating that CSP could promote the apoptosis of HepG2.3 NLS of CSP inhibited the activation of NF-κB through outcompeting nuclear translocation of NF-κB（1） CSP inhibited the activation of NF-κB of tumor cells by its NLS motif: NF-κB reporter gene assay showed that both pFLAG-CMV8-CSP and pFLAG-CMV8-CSP NLS, but not pFLAG-CMV8-CSPΔNLS, could inhibit the activation of NF-κB in both two tumor cells at a comparable level.（2） CSP competitively inhibited the nuclear translocation of NF-κB of tumor cellsEMSA showed that the bands of NF-κB of tumor cells transfected with recombinant plasmid pFLAG-CMV8-CSP were markedly reduced, compared to the control transfected with pFLAG-CMV8, suggesting that CSP could inhibit the nuclear translocation of NF-κB in both SW480 and HepG2.In summary, our data demonstrated that Plasmodium yoelii BY265 CSP could suppress the growth of human colon cancer cell SW480 through inhibition of NF-κB activation, and NLS domain plays an important role by outcompeting nuclear translocation. This could provide new sight for further research on influence of CSP on the migration of tumor cells and tumor biological therapy.

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