Bax and APPL1 are involved in DCC-ADD induced colorectal ...

More documents

Recommendations

Info

III. Discussion The pro-apoptotic activity of DCC-ADD has long been discussed. In the present study, we constructed the recombinant plasmid pIRES2-EGFP-ADD, and transfected it into human colorectal cancer cells SW1116. The results from precise methods of CCK-8 cell proliferation assay, AO/EB fluorescence staining assay and TUNEL assay confirm that point once again. The possible mechanism of DCC-induced apoptosis is related to the cleavage of DCC at Asp1290 by caspase3 leading to the exposure of ADD, which activates caspase9 and the downstream apoptotic signals (Mehlen P, et al., 1998). Bax is a member of the Bcl-2 family and plays a central role in the mitochondria-dependent apoptotic pathway. When receiving a death signal, Bax can promote the release of cytochrom c from mitochondria, which can combine with Apaf-1 and activate caspase9 (Lalier L, et al., 2007; Renault TT, et al., 2011). Our datas revealed that the Bax expression of colorectal carcinoma cells was increased obviously post DCC-ADD gene transfection, which gives new clues that DCC may induce apoptosis through the mitochondria-dependent apoptotic pathway. Gene Therapy and Molecular Biology Vol 14, page 59 59 It has been demonstrated that APPL1 (also called DCC-interacting protein-13, DIP13) can mediate the proapoptotic activity of DCC through the interaction between its DCC-interacting domain and DCC-ADD. Co-expression of DCC and APPL1 can increase DCC induced apoptosis obviously. And removal of the DCC-interacting domain on APPL1 or inhibition of APPL1 expression blocks DCCinduced apoptosis (Liu J, et al., 2002). In the present study, the result showed that DCC- ADD gene transfection induced colorectal carcinoma cells apoptosis and enhanced the expression level of APPL1. ADD can interact with initiator of caspase9 and promote the activation of caspases leading to cell apoptosis (Mehlen P, et al., 1998; Forcet C, et al., 2001). As the adapter protein between ADD and caspase9, APPL1 plays an important role in DCC-induced apoptosis (Liu J, et al., 2002). In summary, we surmise the pathway of DCC inducing apoptosis may due to its ADD being exposed by cleavage of caspase3. And Bax and APPL1 mediate the activating of caspase9 by ADD, which promotes the apoptosis cascade at last. Among them, the expression and activity changes of the caspase9 need further experimental validation.
Cong et al: Bax and APPL1 are involved in DCC-ADD induced colorectal carcinoma cells apoptosis Figure 1: ADD gene transfection inhibits SW1116 cells proliferation and induces apoptosis. (a) Obtaining ADD-coding sequence and constructing recombinant plasmid pIRES2-EGFP-ADD. Lane 1 and 2: ADDcoding sequence obtained by denaturating and annealing of its sense strand and anti-sense strand oligodeoxynucleotides; lane 3: restriction enzyme XhoI and NdeI-digested recombinant plasmid pIRES2-EGFP-ADD. (b) Plasmid pIRES2-EGFP-ADD transfection inhibits SW1116 cells proliferation. Cell proliferation was detected through CCK-8 cell proliferation assay at 36 h post transfection. Each symbol represents the mean OD values ± SD of cell proliferation. (c) Plasmid pIRES2-EGFP-ADD transfection induces SW1116 cells apoptosis detected through AO/EB staining assay at 24 h post transfection. The arrows indicate the apoptotic cells in red fluorescence. Magnification, ×200. (d) Plasmid pIRES2-EGFP-ADD transfection induces SW1116 cells apoptosis detected through TUNEL assay at 24 h post transfection. The arrows indicate the brown-stained apoptotic cells. Magnification, ×200. Figure 2: Bax and APPL1 expression increases in ADD gene-transfected SW1116 cells. (a) Bax and APPL1 expression detected by immunocytochemical method at 20 h post plasmid pIRES2-EGFP-ADD transfection. The arrows indicate the brown-stained positive cells. Magnification, ×200. (b) Bax expression detected by flow cytometry at 20 h post transfection. 60
Page 1 and 2: Gene Therapy and Molecular Biology
Page 3: This method was based on the differ

Bax and APPL1 are involved in DCC-ADD induced colorectal ...

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?