1. Volume 17, Issue 9, Pages 1509-1516 (September 2009)
BH3-based Fusion Artificial Peptide Induces Apoptosis and Targets Human Colon Cancer 
Yongjun Liu, Yunfeng Li, Haijuan Wang, Jing Yu, Hongwei Lin, Dongkui Xu, Yang Wang, Ailing Liang, Xiao Liang, Xueyan Zhang, Ming Fu, Haili Qian, Chen Lin 
Molecular Therapy 
Volume 17, Issue 9, Pages (September 2009)
DOI: /mt
Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

2. Figure 1
Fusion peptides penetrate cells and inhibit cell growth. (a) Schematic structure of peptides used in the study. (b) Survival rates of three cell lines cocultured with 60 µmol/l BH3, TAT-DV3, or TAT-DV3-BH3 for 72 hours. The survival rates were compared with analysis of variance. Results indicated that BH3 and TAT-DV3 had no effects on these three cell lines (P > 0.05). Only TAT-DV3-BH3 inhibited the growth of HCT116p53+/+ and HCT116p53–/– significantly (P < 0.01), but not of HEK293 (P > 0.05). The effects of TAT-DV3-BH3 on two colon cancer cells and HEK293 are different significantly (P < 0.01). (c) Distribution of TAT-DV3-BH3 in cells. The green color shows the fluorescein isothiocyanate-labeled peptides and the red color shows the mitochondria stained with Mito Tracker Red CMXRos. The yellow color represents the colocalization of peptides and mitochondria. Scale bars stand for 10 µm. (d) Histogram of peptides transfection efficiency after cocultured for 0.5 hour with HCT116p53–/–, HCT116p53+/+, and HEK-293. The three cell lines were high-efficiently transfected with TAT-DV3-BH3. TAT-DV3 also labeled HCT116p53–/– cells, indicating it is able to penetrate cell membrane. Although BH3 also labels HCT116p53–/– cells, but its efficiency is much less than that of TAT-DV3-BH3. BH3, Bcl-2 Homology 3; TAT, trans-activator of transcription.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

3. Figure 2
Apoptosis rate was obtained by flow cytometry analysis after cells were cocultured with various polypeptides for 72 hours, followed by 70% alcohol fixation. (a) The histogram of apoptosis in different conditions. A–F stand for HCT116p53+/+, G–L for HCT116p53–/–, and M–R for HEK293. (b) Bars of apoptosis rate. BH3, Bcl-2 Homology 3; TAT, trans-activator of transcription.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

4. Figure 3
Apoptosis assay after peptide treatment. (a) Apoptotic nuclear changes of colon cancer cell HCT116p53–/– under fluorescence microscope after treatment with 60 µmol/l TAT-DV3-BH3 and 4,6-diamidino-2-phenylindole dihydrochloride staining. After 48 hours of treatment, nuclei showed characteristic crimpled nuclear membrane, condensed chromatin. After 72 hours of treatment, the nucleuses exhibited remarkable membrane pycnosis, chromatin condensation and fragmentation. All of these signs are representative of apoptosis. (b) Detection of key molecules in apoptosis activation 72 hours after TAT-DV3-BH3 treatment. The proenzyme of caspase-9 was reduced and its activated form accumulated with dose increase, while neither proenzyme nor activated form of caspase-8 showed any significant change. Procaspase-3 displayed a decrease by TAT-DV3-BH3 treatment. Actin was detected as loading control. BH3, Bcl-2 Homology 3; TAT, trans-activator of transcription.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

5. Figure 4
Fusion peptides suppress tumor growth in vivo. (a) Body weight of mice bearing tumor increase gradually. Mice grew in a normal health condition, showing no obvious toxic effects of peptides. There is no significant difference between these four groups in body weight (P > 0.05). (b) Growth of tumor volumes in different treatment groups. Tumor volumes are measured every 2 days after Scatter of volume of tumor. Zero stands for volume before first injection and injection from the 1st day to 7th day. The time of weight is before injection. There was significance between TAT-DV3-BH3 group and the others from the 12th day, P < (c) Tumor weight of each group. There is significant difference between TAT-DV3-BH3 and other groups (P < 0.05), but no difference between TAT-DV3, BH3, and control groups (P > 0.05). BH3, Bcl-2 Homology 3; TAT, trans-activator of transcription.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

6. Figure 5
Distribution of peptides in different organs after tail-rein injection. (a) The tumors and organs from mice models injected with polypeptides and PBS 3 hours before detection. The tumors and organs were resected and aligned as indicated to be detected for peptide condensation. The tumors and organs were detected under in vivo imaging system. (b) Tumors from TAT-DV3-BH3, TAT-DV3, and BH3 groups displayed green fluorescence signal. Only liver and kidney from peptide injection group showed weak fluorescence signal, which may indicate the metabolism or secretion of peptides by these organs. Bar = 5 mm. BH3, Bcl-2 Homology 3; PBS, phosphate-buffered saline; TAT, trans-activator of transcription.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions