Molecular Mechanism And Effect Of Calcyclin Binding Protein (CacyBP) On Gastric Cancer Development

The maintaining of the cytoplastic Ca²⁺steady and the calcium signaling is of importance of the life activities. Ca²⁺ Binding Protein is the key molecule in controlling the Ca²⁺ balance and signaling transduction. S100 family is the biggest subfamily in CaBP, which play its role by binding to its target proteins. Some research revealed that the close relationship between the S100 and carcinogenesis and metastasis with unknown mechanisms. CacyBP is the target protein of S100 found recently, with MW of 30KDa and 229 amino acids. To date, it mainly expresses in neural tissues and binds to some members in S100 family in a Ca²⁺ - dependent way. Also, the Ca²⁺-dependent translocation and phosphorylation of CacyBP within the neural cell is reported. CacyBP/SIP, a component of a novel ubiquitinylation complex leading to β -catenin degradation, which might involve in the tumorigenesis and progression. It is found by our previous work that CacyBP is upregulated in the drug-resistant gastric cancer cells, with the translocation within the cells. That indicates its involvement in gastric carcinogenesis. This work aims to elucidate the role and mechanism of CacyBP in gastric cancer and try to find the relationship of the significance of its translocation and phosphorylation. Thatwill help in understanding the functionality of CacyBP and S100-CacyBP mediated Ca²⁺ signaling transduction and contribute to the well-illustration of the tumorigenesis.[Purpose] 1. To elucidate the mechanism of CacyBP translocation and phosphorylation within the gastric cancer cell; 2. To study the relation between CacyBP phosphorylation and its function of protein degradation; 3. To understand the effect of CacyBP on the gastric cancer cell and its mechanism[Methods] 1. By indirect immunofluorescence, find out the location of CacyBP in the immortalized gastric epithelial cell lines GES, gastric cancer cell SGC7901 and SGC7901/ADR; dectect the Ca²⁺ concentration by Laser Scanning Confocal microscope loaded with FIuo-3/AM. 2. treat SGC7901 with different concentration of ionomycin and detect the translocation of CacyBP by Ca²⁺ alteration; check the phosphorylation of CacyBP and the expression of β - catenin by Ca²⁺+ alteration with coimmuno-precipitation and immunoblot technics. 3.observe the translocation of CacyBP by the alteration of PKC activity and detect phophorylation of CacyBP and expression of β — catenin. 4. understand the translocation and phosphorylation of CacyBP in different cell cycle by synchronizing through double thymidine blockage. 5. construct the NLS(nucleotide localization signals)-null mutation of CacyBP and transfect into cell; observe the translocation of CacyBP by Ca²⁺alteration; understand the significance of NLS in its translocation. 6. construct the sense and RNAi expression vector of CacyBP, transfect into SGC7901 and select stable clones by G418 screening. 7. perform RT-PCR, western blot and indirect immunofluoresence on different stable clones to identify the transfection. 8. depict the growth curves of the transfectants and the control cells.9. Flow CytoMeter( FCM) is used to identify the cell cycle distribution of these cells. 10.cloning formation assay on plate and soft agar is applied to test the tumorigenesis in vitro of the two cells.ll. tumor xenograft in nude mice is performed to test the in vivo tumorigenesisof the cells. 12. Transwell assay is used to test the invasion ability in vitro.13. identify the expression of the apoptosis associated molecules (Fas, p53, bax, bcl-2 and cytochrome C)by RT-PCR and western blot. 14. primary identification of CacyBP target molecules by RT-PCR and western blot.[Results] 1. It was showed by immunofluorescent staining that CacyBP distributed in the cytoplasm of GES and SGC7901, while in both of the cytoplasm and nucleus of SGC7901/ADR with the dominant perinuclear distribution. The concentrations of free Ca²⁺ were increased in GES, SGC7901 and SGC7901/ADR accordingly. 2. The concentration of free Ca²⁺ will induction by ionomycin in a dose-dependent way. The nuclear translocation of CacyBP can be seen with increasing of Ca²⁺ and more increase of Ca²⁺can, however, lead to the perinuclear distribution of CacyBP again. Treated by ionomycin on SGC7901, both the total and phosphorylated protein of CacyBP were increased markedly, while the expression of β—catenin decreased according to the increase of ionomycin.3. The nuclear and perinuclear translocation of CacyBP was happened after 2 hours of PKC activator PMA (10nM, 50 nM å’Œ 100 nM) induction, while it was not happened when it is PMA simulant 4 α — PMA instead of PMA. The concentration of Ca²⁺ was not altered by adding PMA. It is concluded that PKC can directly induce the translocation of CacyBP, without the aleration of Ca²⁺. 4. After being synchronized, CacyBP was found to distribute mainly in cytoplasm in G1 and S phase, while it was perinuclear and nuclear translocated when in G2 phase, which leading to the conclusion that the translocation was cell phase dependent. The total protein of CacyBP was not changed in all phases, while the phosphorylated protein was elevated in G2 phase, with the concomitantly decreased expression of β —catenin. It comes to the conclusion that some molecule in G2 phase might play its role in the translocation and phosphorylation of CacyBP. 5. The NLS-null mutation vector was successfullyconstructed and transfected into SGC7901 cells. The purpose protein were found in transfeced cells detected by anti-FLAG antibody. The ectopic CacyBP distributed in nucleus of NLS-null mutant cells treated with ionomycin. 6. The sense and RNAi expression vector of CacyBP were constructed and transfected into SGC7901 and stable clones were selected and identified. 7. The growth of the sense transfectants were slowed down while the RNAi transfectants were accelerated, showed by MTT assay. 8. The FCM indicated that the apoptosis peak were so markable in sense transfectants without the alteration of proliferation index; the G1/S transition was accelerated in RNAi transfectants with the increased proliferation index. 9. The cloning formation assay showed that the RNAi transfectants clone formation rate was higher than the control cells while the sense transfectants were lower than the control cells.10. The Transwell assay indicated that the number of the invasive cells through the membrane were much more in RNAi transfectants than in sense transfectants. 11. Western blot revealed that P53 was increased in sense transfectants, the Bcl-2 and the Bcl-2/bax ratio decreased; while P53 decreased and Bcl-2, Bcl-2/bax ratio increased in RNAi transfectants. Other apoptosis associated molecules, such as Fas, bax and Cyto C were not affected by the CacyBP. Semi-quantitative RT-PCR showed that P53 mRNA increased, bcl-2 mRNA decreased in sense transfectants, while the reverse phenomenon were observed in RNAi transfectants. 12. The other molecules were also detected in order to identify the target molecules of CacyBP. Western blot and semi-quantitative RT-PCR indicated that in sense transfectants, β — catenin, COX-2 ,Hsp7(Kracl and Cyclin D1 were decreased at protein level; COX-2 and Cyclin Dl mRNA were decreased. In the RNAi transfectants, β — catenim COX-2, Hsp70 and Cyclin Dl were increased at protein level; COX-2 and Cyclin Dl mRNA were increased. The β —catenin , racland Hsp70 mRNA were not changed in both transfectants, neither did protein and mRNA of Raf-1.