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. 2009 Apr 7;17(9):1509–1516. doi: 10.1038/mt.2009.43

BH3-based Fusion Artificial Peptide Induces Apoptosis and Targets Human Colon Cancer

Yongjun Liu 1,2, Yunfeng Li 1, Haijuan Wang 1, Jing Yu 1, Hongwei Lin 1, Dongkui Xu 1, Yang Wang 1, Ailing Liang 3, Xiao Liang 1, Xueyan Zhang 1, Ming Fu 1, Haili Qian 1, Chen Lin 1
PMCID: PMC2835260  PMID: 19352325

Abstract

Dysregulation of apoptosis is a pilot event before cancer development and plays important roles for cancer to develop resistance to chemical therapeutics. So exploring strategies to recovery the apoptosis balance is a charming and long-endeavored aim in the attempts to conquer cancers. The present study shows an exciting potency of a fusion peptide to inhibit and target to cancer cells, which is composed of BH3 (Bcl-2 Homology 3) effector domain from PUMA (p53 upregulated modulator of apoptosis) and targeting domain of trans-activator of transcription (TAT) and DV3. The in vitro results demonstrated cancer growth inhibition by the fusion peptide in colon cancer cells, as well as in lung adenocarcinoma cell line and breast carcinoma cell line of human origin. But the viability of HEK293, a noncancerous cell line, was not affected, indicating the cancer specificity of the fusion peptide. Apoptosis activation was induced by the peptide through the mitochondria pathway. In vivo studies displayed its tumor inhibiting ability by intratumoral injection. When the fusion peptide was administered systematically by tail vein, the peptide targeted the established tumors in nude mice. No other organs were significantly involved. The fusion peptide is an artificially designed molecule worthy of further evaluation and development.

Introduction

Apoptosis or programmed cell death is an essential process in cell homeostasis and eukaryotic development. Failure to appropriately activate the apoptotic program is associated with inflammatory and autoimmune disorders, while premature acceleration of apoptosis has been linked to neurodegenerative disease.1 Apoptosis has undergone extensive scrutiny as a potential target for cancer therapy during the past 30 years. Failure of tumor cells to undergo apoptosis translates into malignant potential and chemotherapeutic resistance.2

PUMA (p53 upregulated modulator of apoptosis) is a “BH3-only” member of the Bcl-2 family that was initially identified by differential gene expression studies as a p53 target gene and a potent inducer of apoptosis. PUMA is normally expressed at low levels in human tissues but can be induced by p53 or DNA damaging agents.3,4 The proapoptotic capacity of PUMA is dependent on its Bcl-2 Homology 3 (BH3) domain, whose conservative sequence comprises nine amino acids (LRRMADDLN).5

Our previous study had demonstrated that adenoviral delivery of PUMA can effectively kill various cancer cells and sensitize esophageal cancer cells to chemotherapeutic agents. For example, Ad-PUMA could reduce the IC50 of chemotherapeutic agents in four esophageal cancer cells. Then, exogenously expressing PUMA or introducing its functional components into cancer cell provides another opportunity for gene therapy and multimodality therapy against cancers.6 Adenoviral delivery of genes against cancer was faced with many obstacles, such as the targeting ability and safety issue. Previous studies have suggested that the BH3 domain is an important active player in PUMA-promoted apoptosis, because deletion of BH3 absolutely castrated PUMA function. So manipulating the BH3 peptide may be another way to circumvent the vector issues for gene transfer.

Peptide drug is a new field of drug development with the advantages of lower molecular weight, lower toxicity, and higher bioavailability. But problems in tumor specificity and in vivo transduction efficiency also exist. The C-X-C motif receptor 4 chemokine receptor is overexpressed on at least 23 types of cancers, including breast cancer, ovarian cancer, glioma, pancreatic cancer, prostate cancer, acute-myelogenous leukemia, etc.7,8 DV3 is the binding domain of C-X-C motif receptor 4 ligand, providing its potential as a tool to target cancer cells in drug design. The transactivator of transcription (TAT) protein of human immunodeficiency virus 1 has been shown to deliver various biological molecules into cell with extremely high efficiency. It can transfer proteins, DNA, RNA, even nanoparticles into cytoplasm in a time as short as 10 minutes with a proximate 100% of efficiency.9 In this study, we linked DV3 and TAT domains to BH3 as a “helper” to facilitate the core peptide to target and penetrate the cancer cells. The cancer suppression effects and cancer-targeting efficiency were evaluated in vitro and in vivo to support further development.

Results

TAT-DV3-BH3, but not other shorter control peptides, inhibited the proliferation of colon cancer cells but not HEK293 cells

We chemically synthesized short peptides TAT-DV3-BH3, TAT-DV3, and BH3 to confirm the suppressive effects of these peptides on cancer cells (Figure 1a). Results indicated that neither BH3 nor TAT-DV3 exerted inhibition on growth of three selected cell lines, while TAT-DV3-BH3 inhibited cancer growth significantly (Figure 1b). Interestingly, the TAT-DV3-BH3 did not inhibit the growth of HEK293 cell line. The survival rates of these three cell lines in TAT-DV3 and BH3 treated groups ranged from 89 to 112% (P > 0.05). However, the survival rates of two colon cancer cells by TAT-DV3-BH3 treatment were 33.29 ± 4.13% (HCT116p53+/+) and 31.53 ± 0.08% (HCT116p53–/–) (P < 0.01). This suggested the specificity of TAT-DV3-BH3 to cancer cells.

Figure 1.

Figure 1

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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.

TAT-DV3-BH3 significantly inhibited the growth of a panel of cancer cells

Next, we tested the potential cytotoxicity of TAT-DV3-BH3 in a series of cell lines by measuring the viability of cells with CCK-8 kit. The results indicated that the proliferation of a wide spectrum of cancer cells was inhibited by TAT-DV3-BH3. The colon cancer cells were much more sensitive to TAT-DV3-BH3 than other tested cancer cell lines (P < 0.01). The survival rates of two colon cancer cell lines, HCT116p53+/+ and HCT116p53–/– are 32.19% and 34.28% at indicated peptide concentration, respectively. Also, TAT-DV3-BH3 inhibited proliferation of lung adenocarcinoma cell line GLC-82 and breast cancer cell line MDA-MB-231 significantly (P < 0.05), with cell survival rates of 63.64 and 69.79%, respectively. For the two cell lines of normal origin, HEK293 and 2BS, which are human embryonic kidney and lung origins, TAT-DV3-BH3 showed no inhibition on cell growth (Table 1). We chose HCT116p53+/+, HCT116p53–/–, and HEK293 for further evaluation of TAT-DV3-BH3 activity, while HEK293 was taken as control.

Table 1.

Survival rates of cells treated with 60 µmol/l TAT-DV3-BH3 for 72 hours

graphic file with name mt200943t1.jpg

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TAT-DV3-BH3 was distributed mainly in mitochondria area

For PUMA plays its role by binding to mitochondria-anchored molecules, we supposed that TAT-DV3-BH3 would take effects by involving mitochondria. We labeled the synthesized peptide with fluorescein isothiocyanate (FITC) to trace its distribution in cells. When the treated cells were investigated under confocal microscopy after 0.5 hour of incubation, the peptide was located on cell membrane. With 1 hour of incubation, it was located in cytoplasm, but not in nuclear. 24 hours of incubation did not change the distribution pattern. After the cells were stained with mitochondria-specific dye Mito Tracker Red CMXRos, it was found that the TAT-DV3-BH3 mainly overlapped with mitochondria area in the cytoplasm. Compared with TAT-DV3-BH3, though the TAT-DV3 and BH3 also entered into cytoplasm, they were not concentrated in the same manner in the mitochondria area (Figure 1c).

TAT-DV3-BH3 entered into cells with high efficiency

We monitored the efficiency of TAT-DV3-BH3 to enter into cancer cells by fluorescence-activated cell sorter. By FITC-labeling, peptides were sorted with fluorescence-activated cell sorter. Both TAT-DV3-BH3 and TAT-DV3 transfected HCT116p53–/– with the efficiency of 99.5 and 94.4%, which were much >58.3% of BH3. In HCT116p53+/+ and HEK293, the labeling rates of TAT-DV3-BH3 were 99.9 and 99.9%, respectively (Figure 1d). All of the peptides shared the ability to penetrate cells with various extends, though not all of them exerted effects on cell growth.

TAT-DV3-BH3 induced apoptosis of cancer cell lines but not HEK293

Although TAT-DV3-BH3 impressively inhibited the growth of cancer cells, we do not know whether the fused polypeptide induced apoptosis, which is a main biological effect of PUMA. The results from flow cytometry indicated that the apoptosis rate of colon cancer cells increased by the fused polypeptide in a concentration-dependent manner. But in the HEK293 cell line, there was no significant difference for the TAT-DV3-BH3 peptide, with an apoptosis rate similar to that of control group (P > 0.05, Figure 2). The two control peptides, TAT-DV3 and BH3, showed no proapoptotic effects on cancer cell lines and HEK293 cells. These results suggested that only TAT-DV3-BH3 had the potency and specificity to promote apoptosis in cancer cells.

Figure 2.

Figure 2

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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.

TAT-DV3-BH3 induced colon cancer cells to exhibit apoptotic morphology

4,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining showed apoptotic morphology of colon cancer cells after the cells were cocultured with 60 µmol/l TAT-DV3-BH3 for 48 hours. When cancer cells were treated with TAT-DV3-BH3 for 72 hours, cells showed typical apoptotic nuclear changes (Figure 3a). Neither colon cancer cells nor HEK293 showed apoptotic morphology by TAT-DV3 or BH3 treatment (data not shown).

Figure 3.

Figure 3

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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.

TAT-DV3-BH3 induced apoptosis of colon cancer cells through mitochondria pathway

To explore the mechanism by which fused polypeptide induced cancer cell apoptosis, we detected the expression of key molecules involved in cell apoptosis by Western blot. Generally, there are two apoptosis pathways that involve caspase-9 and caspase-8, respectively. Caspase-9 activation indicates mitochondria pathway and caspase-8 activation means the death receptor pathway. By TAT-DV3-BH3 treatment, caspase-9 was activated with procaspase-9 reduction in a dose-dependent manner (Figure 3b). But for the death receptor pathway player caspase-8, it was not activated significantly. The procaspase-3 also showed a decreasing tendency when dose increased, which may have resulted from its activation. These results indicated that the fused polypeptide induced cancer cell apoptosis via mitochondria pathway (Figure 3b). We did not detected significant activation of apoptosis pathway by TAT-DV3 or BH3 in cancer cell, not even in HEK293 cells (data not shown).

Fused polypeptide slowed down the growth of tumors in nude mice model

To investigate the in vivo effects of fused peptides on tumor, we established nude mouse model bearing HCT116p53–/– tumor. The mice lived in a normal healthy condition during peptides treatment by introtumoral injection, gaining weight gradually similar to the control group (normal saline was given) (P > 0.05, Figure 4a). The tumor volumes in TAT-DV3-BH3-treated group were much lower than those of the other groups. From day 12, the difference between TAT-DV3-BH3 group and other groups became statistically significant (P < 0.05, Figure 4b). There was no significant difference between TAT-DV3, BH3, and saline groups (P > 0.05, Figure 4b). On day 20, the mice were sacrificed, the tumor weight in TAT-DV3-BH3 group were significantly much lower than that of TAT-DV3, BH3, and saline groups, while there was no statistically significant difference between these three groups (Figure 4c). The inhibition rates of TAT-DV3-BH3, TAT-DV3, and BH3 on tumor growth are 56.9, 14.8, and –29.3%, respectively.

Figure 4.

Figure 4

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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 < 0.05. (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.

Systematic distribution of polypeptides in tumor-bearing nude mice demonstrated in vivo tumor-specific targeting

To evaluate the in vivo specificity of the fused peptides, we established HCT116p53–/– tumor models in nude mice. The polypeptides as well as the phosphate-buffered saline (PBS) as control were injected into mice through the tail vein 3 hours before in vivo imaging screening. We sacrificed the mice and resected the tumors to observe them under in vivo imaging system. Also, the organs of heart, lung, liver, kidney, and spleen were detected under the imaging system. Green fluorescence was observed in tumors of TAT-DV3-BH3, TAT-DV3, and BH3 groups (Figure 5a,b). In the studied organs and tissues, only liver and kidney showed weak green fluorescence signal, which might have come from the metabolism of the peptides in the body. It is totally negative in other organs and tissues. Tumors and organs from PBS group were negative in fluorescence signal.

Figure 5.

Figure 5

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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.

Discussion

Dysregulation of apoptosis is a prerequisite for the development of cancer and one of the main reasons for drug resistance of cancer. Biotherapy became the fourth therapy mode for cancer since Anderson et al.10 first succeeded in curing one girl with severe combined immunodeficiency by using the gene of adenine nucleotide deaminase in 1990.

PUMA is a potent molecule to induce cancer cell apoptosis. It was shown that exogenous expression of PUMA resulted in a rapid and complete apoptosis in a variety of cancer cell lines.4,9,11,12 Intriguingly, morphological signs of apoptosis, such as condensed chromatin and fragmented nuclei, appeared ~9 hours later in DLD1 cells induced by p53 expression than those induced by PUMA expression.4 At a very low titers of adenoviral vector carrying PUMA coding sequence at which only <10% cancer cells get affected, PUMA sensitizes cancer cells to various anticancer drugs, such as cDDP, PXT, and 5-Fu.6,13 The greater power of PUMA than p53 in inducing apoptosis makes it a first-class choice in the attempts of cancer biotherapy.14 It completely loses its apoptosis-promoting potency once the core BH3 domain is deleted, indicating an irreplaceable role of BH3 in its natural function.5,15

It is necessary to map the minimal functional fragment of PUMA to increase the flexibility in future application. In this study, we investigated the effects of multiple functional fused polypeptides, based on BH3 domain of PUMA on cancer cells in vitro and in vivo, primarily exploring the feasibility of our designed fused polypeptides to treat cancer. Results showed that BH3-based fusion polypeptide effectively induces cancer cell apoptosis through mitochondria pathway and inhibits cancer growth in vivo. Most interestingly, it spatially and functionally targets cancer cells both in vitro and in vivo. The specificity to cancer cells endows a great interest in our designed peptide in future development. The fused polypeptide TAT-DV3-BH3 exerted significant inhibition on proliferation of colon cancer cells HCT116p53–/–, but did no harm to HEK-293. The TAT-DV3 subunit in the TAT-DV3-BH3 is designed to combinationally enhance the specificity of BH3 subunit to cancer cells with high efficiency of transmembrane delivery. In the current study, neither TAT-DV3 nor BH3 alone has the power to suppress cancer cells, though they have some extend of ability to enter into cells, indicating the necessity of BH3 to enter into cytoplasm for function. Though we did not respectively evaluate the individual contribution of TAT and DV3 to the specific targeting of TAT-DV3-BH3, the fact that BH3 alone took no effects on cancer cells is a strong support for the subunit's comprehensive contribution. Based on the high efficient delivery of TAT and high frequency of C-X-C motif receptor 4 upregulation in many types of cancers, it is reasonable to expect that the TAT-DV3 platform bears universal application in transferring peptides into cancer cells.7,8

In our study, when combined with TAT-DV3, BH3 as a core functional domain of PUMA shares the same mechanism as natural PUMA molecule in inducing cancer cell apoptosis through mitochondria pathway.16,17,18 PUMA activates caspase-9 and caspase-3 rapidly in colorectal cancer cell lines,4 which is consistent with our results.

In the in vivo experimental treatment of cancer by TAT-DV3-BH3, TAT-DV3, and BH3, only the entire form of TAT-DV3-BH3 exhibited cancer inhibition effects, while TAT-DV3 and BH3 have no suppressive effects on cancer in vivo. These facts are consistent with the data from in vitro studies in which only TAT-DV3-BH3 was effective in promoting cancer cell apoptosis and growth suppression. It is exciting to see the in vivo tumor-targeting of TAT-DV3-BH3. Three hours after systematic administration, TAT-DV3-BH3 as well as TAT-DV3 distributed mainly to tumor regions, but not other organs, except liver and kidney, exhibited peptides accumulation visualized by green fluorescence of FITC, indicating high specificity of peptides to cancer cells in vivo. The pharmacokinetic parameters will be studied in detail in the future. Interestingly, we also observed the BH3 distribution in tumors in a manner similar with TAT-DV3-BH3 and TAT-DV3, though it showed no suppressive effects on tumor growth in vivo. In the in vitro studies, we also observed the binding and cytoplasm delivery of BH3 to cancer cells and HEK-293 cells. The mechanism about how the net BH3 domain was delivered into cells but without noticeable biological activity is remain unknown. It has been reported that the minimal fragment of BH3 domain has no biological activities.5 We suppose that additional flank sequence may be needed for BH3 function to form more active structure. Optimization of the flank sequence may help enhance the potency of BH3 domain. Another advantage for BH3-based peptide in cancer treatment may be its inability to induce immunoresponse. There is no detectable antibody production in the serum during the 28 days of experimental treatment (data not shown).

Here we provide a highly cancer-selective and potent fused polypeptide based on TAT facilitated transfer and DV3-guided targeting, as well as PUMA BH3 effector. Though there are more details to be clarified, it is worthy of further evaluation and development in cancer treatment.

Materials and Methods

Polypeptide synthesis. The peptides used in the study were chemically synthesized by the Genetimes Technology (Shanghai, China). Three peptides were synthesized with >95% purity. The peptide TAT-DV3-BH3 was used as the expected functional molecule with sequence of YGRKKRRQRRRGGGLGASWHRPDKGGGGLRRMADDLNAQY; TAT-DV3 (YGRKKRRQRRRGGGLGASWHRPDKG), and BH3 (LRRM ADDLNAQY) were also produced as controls. Between two fragments, three glycines were inserted to enhance the plasticity. FITC was conjugated to the end of the peptide for the feasibility of localization. These peptides were dissolved in sterile water for use.

Cell culture. Human colon cancer cell lines HCT116p53+/+ and HCT116p53–/– were maintained in McCoy's 5A media, human embryonic kidney fibroblast HEK-293,19 human adenocarcinoma of lung cell line GLC-82,20 and human breast cancer cell line MDA-MB-23121 were maintained in Dulbecco's modified Eagle's medium, and human gastric cancer cell line SGC-790122 was cultured in RPMI 1640 medium. All media were supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. All cell-culture reagents were obtained from Gibco (Invitrogen, Paisley, UK). Cells were cultured in a 5% CO2 incubator at 37 °C with humidified air.

Cytotoxicity assay of TAT-DV3-BH3 to cell lines. To make sure that these peptides are biologically active, cell survival after TAT-DV3-BH3 treatment was determined by assaying viable cell numbers using the Cell Counting Kit-8 (CCK-8) (cat. no. CK04-13; Dojindo Molecular Technologies, Kumamoto, Japan) according to the manufacturer's protocol.23 The peptide concentration used in the experiment was 60 µmol/l of TAT-DV3-BH3 per well (200 µl/well). After 72 hours of peptide incubation, cell viability was determined by CCK-8 kit according to manufacture's manual. The absorbance at 450 and 630 nm was read using the Microplate Reader (Bio-Rad 680; Bio-Rad, Hercules, CA). The experiment was repeated in triplicate. Survival Rate (%) = (Atreated – Ablank) × 100 / (Acontrol – Ablank).

Cytotoxicity assay for TAT-DV3, BH3, and TAT-DV3-BH3. To compare the inhibitory effects of TAT-DV3-BH3, TAT-DV3, and BH3, cytotoxicity assay was performed as dictated above. The concentration of TAT-DV3-BH3, TAT-DV3, and BH3 were 60 µmol/l.

Location of polypeptides in cells. The colon cancer cell line HCT116 and HEK293 were, respectively, cultured in 35 mm plates with slide cover on the plate bottom with TAT-DV3-BH3 at 40 µmol/l for 0.5 hour, 1 hour, and 24 hours, respectively; HCT116p53–/– was cocultured with TAT-DV3, BH3, and TAT-DV3-BH3 at 40 µmol/l for 0.5 hour, 1 hour, and 24 hours, respectively. And then, these cells were stained with mitochondria dye Mito Tracker Red CMXRos (cat. no. M7512; Invitrogen, Carlsbad, CA) as the manufacturer's protocol.24 After staining, cells were washed with fresh, prewarmed growth medium for three times. Then the medium were removed and cells were incubated at 37 °C for 15 minutes with prewarmed growth medium containing 4% methanol. Cells were rinsed three times with PBS, and the slide covers were covered onto the slide. All slides were observed at the laser scanning confocal microscopy (Leica TCS SP2; Leica Microsystem, Wetzlar, Germany).

Flow cytometry detecting transfection efficiency of fused peptides. To evaluate the transfection efficiency of fused polypeptides, HCT116p53–/–, HCT116p53+/+, and HEK293 were cocultured with 40 µmol/l fused polypeptide for 0.5 hour, and then the green fluorescence of the fused polypeptide was analyzed by a fluorescence-activated cell sorter (Beckman Coulter EPICS XL; Beckman Coulter, Miami, FL), using SYSTEM II 3.0 software. The percentages of transfected cells in each population were determined from at least 5 × 103 cells. The control cells were not treated with fused polypeptide.1

Flow cytometry measurement of apoptosis. Cells were cultured in 24-well plate with 6 × 104 cells per well for 24 hours. The medium was discarded and replaced with fresh culture medium containing 15, 30, and 60 µmol/l TAT-DV3-BH3, respectively. After incubation for 72 hours, the attached and floating cells were harvested and fixed with 70% ethanol at 4 °C overnight. After washed twice with PBS, the cells were incubated in RNase A/PBS (100 µg/ml) at 37 °C for 30 minutes. Intracellular DNA was labeled with propidium iodide (50 µg/ml) and analyzed with fluorescence-activated cell sorter, using SYSTEM II 3.0 software. The percentages of sub-G1 cells in each treatment were determined from at least 1 × 104 cells.6,25

DAPI staining to show apoptosis. HCT116p53–/– cells were seeded onto slide covers in 35 mm discs with 1 × 105 cells for 24 hours. The medium was replaced by fresh culture medium containing 60 µmol/l TAT-DV3-BH3. After 0, 48, and 72 hours cells were washed with PBS and fixed in ice-cold methanol for 10 minutes. Then cells were washed again with PBS and stained with 1 µg/ml DAPI for 15 minutes, followed by PBS washing for 5 minutes. Apoptotic cells were morphologically defined by cytoplasmic and nuclear shrinkage and chromatin condensation or fragmentation.25,26

Western blot. Cells were cocultured with 15, 30, and 60 µmol/l TAT-DV3-BH3 for 72 hours, respectively, and harvested. All cells were lysed with lysis buffer [20 mmol/l Tris.Cl (pH 8.0), 137 mmol/l NaCl, 20 mmol/l DTT, 1%NP-40, 2 mmol/l sodium vanadate, 100 µg/ml pheylmethylsulfonyl fluoride, 1 µg/ml aprotinin; Sigma Chemical, St Louis, MO, 10 µl/106 cells] for 40 minutes. Cell lysates were centrifuged with 12,000 rpm at 4 °C for 20 minutes, and the protein concentration was determined. Equal amounts of cellular protein were separated in 10% sodium dodecyl sulfate-polyacrylamide gels and transferred onto nitrocellulose membranes (Bio-Rad). The membranes were, respectively, incubated with the following primary antibodies: caspase-3 (1:500), caspase-8 (1:500) (cat. nos 9668, 9542, 9746; Cell Signaling Technology, Danvers, MA), and caspase-9 (1:500) (cat. no. 8355; Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4 °C, followed by incubation with corresponding secondary antibody (1:2,000) conjugated to horseradish peroxidase. SuperSignal ECL (Applygen Technologic, Peking, China) was used to visualize the antibody binding. To control protein loading, membranes were stripped and reprobed with anti β-actin antibody (1:2,000).19,27

In vivo antitumor effect of TAT-DV3-BH3. All animal experiments were approved by the Institutional Animal Care and Use Committee at the Peking union medical college. Xenograft tumors were established by s.c. injection of 1 × 107 HCT116p53–/– cells28 into right flanks of 13–15 g (5–6 weeks old) female athymic Nu/Nu (NU-Foxn1nu; Charles River Laboratories, Boston, MA) mice (Vitalriver, Peking, China). Treatment of the tumors started 1 week after tumor implantation when their sizes reached 3–5 mm in diameter. Each experimental group contained 7 mice. The mice were treated with TAT, TAT-DV3, TAT-DV3-BH3 (1.2 mmol/l × 100 µl, respectively), and normal saline once a day for 7 days by intratumoral injection respectively. The tumor volumes and weight of nude mice were measured once every 2 days. The tumor volumes and weights were calculated.1

Distribution of fused polypeptide in nude tumor-bearing mice. To elucidate the distribution of fused polypeptide TAT-DV3-BH3 in nude mice bearing tumor, we detected the distribution of FITC-labeled polypeptides 3 hours after administration of polypeptides from tail veins. In vivo Imaging System I.C.E (Roper Scientific, Trenton, NJ) was adopted for the detection. We also took the tumors and organs from nude mice for green fluorescence detection. Nude mice were injected by tail vein with 100 µl volumes of PBS, 1.2 mmol/l BH3, TAT-DV3, or TAT-DV3-BH3, respectively. The animals were anesthetized with 0.15 ml tribromoethyl alcohol/10 g weight before detection of fluorescence signal.

Statistical analysis. Data are presented as means and SD. Statistical differences between two groups were evaluated using the independent-samples t-test. One-way analysis of variance was used for the comparisons among three or more groups. A P-value of <0.05 was considered statistically significant.

Acknowledgments

This work is done in State Key Laboratory of Molecular Oncology, Cancer Institute, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China. We thank Yu Jiang (Pittsburgh University) and Zhang Lin in (Pittsburgh University) for presenting colon cancer cells HCT116p53+/+ and HCT116p53–/–. The project was supported by grants from National Natural Science Foundation of China (No. 30600745) and National Basic Research Program of China (973 Program) (2006CB910102 and 2009CB521801). We are greatly appreciated for the supports.

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