Brought to you by Portland Press Ltd.
Published on behalf of the International Federation for Cell Biology
Cancer Cell death Cell cycle Cytoskeleton Exo/endocytosis Differentiation Division Organelles Signalling Stem cells Trafficking
Cell Biology International (2011) 35, 841–848 (Printed in Great Britain)
RNA interference-mediated silencing of focal adhesion kinase inhibits growth of human malignant glioma xenograft in nude mice
Guan‑Jie Wang1, Yong‑Ping Ma1, Yang Yang, Na Zhang, Wei Wang, Sheng‑Yong Liu, Li‑Juan Chen, Yu Jiang, Xia Zhao, Yu‑Quan Wei and Hong‑Xin Deng2
State Key Laboratory of Biotherapy, West China Hospital and West China Medical School, Sichuan University, Chengdu, Sichuan, Peoples Republic of China

FAK (focal adhesion kinase), which plays a pivotal role in mediating cell proliferation, survival and migration, is frequently overexpressed in human malignant glioma. The expression of FAK increases with the advance of tumour grade and stage. Based on these observations, we hypothesized that attenuation of FAK expression may have inhibitory effects on the growth of malignant glioma. In the present study, human glioma cell line U251 was transfected with plasmids containing U6 promoter-driven shRNAs (small-hairpin RNAs) against human FAK using cationic liposome. The effects of FAK knockdown in U251 cells in vitro were analysed by using flow cytometry and PI (propidium iodide)-staining assays. Based on the encouraging in vitro results with FAK silencing, plasmids encoding FAK-targeted shRNA were encapsulated by DOTAP (dioleoyltrimethylammonium propane):Chol (cholesterol) cationic liposome and injected via tail vein to evaluate its therapeutic efficiency on suppressing tumour growth in a human glioma xenograft model. PCNA (proliferating-cell nuclear antigen), CD34 immunostaining and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) assay were used to assess the changes in tumour angiogenesis, apoptosis and proliferation respectively. The results indicated that DOTAP:Chol cationic liposome could deliver therapeutic plasmids systemically to tumour xenografts, resulting in suppression of tumour growth. Treatment with plasmid encoding FAK-targeted shRNA reduced mean tumour volume by approx. 70% compared with control groups (P<0.05), accompanied with angiogenesis inhibition (P<0.05), tumour cell proliferation suppression (P<0.05) and apoptosis induction (P<0.05). Taken together, our results demonstrated that shRNA-mediated silencing of FAK might be a potential therapeutic approach against human malignant glioma.

Key words: angiogenesis, apoptosis, focal adhesion kinase (FAK), RNA interference (RNAi), small-hairpin RNA (shRNA)

Abbreviations: Chol, cholesterol, DMEM, Dulbecco's modified Eagle's medium, DOTAP, dioleoyltrimethylammonium propane, FAK, focal adhesion kinase, GAPDH, glyceraldehyde-3-phosphate dehydrogenase, GS, glucose solution, MVD, microvessel density, PCNA, proliferating-cell nuclear antigen, PI, propidium iodide, RNAi, RNA interference, shRNA, small-hairpin RNA, siRNA, small interfering RNA, TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling, VEGF, vascular endothelial growth factor

1These authors contributed equally to this work.

2To whom correspondence should be addressed (email:

1. Introduction

Malignant glioma represents one of the most lethal and angiogenic cancers. Despite advances in modern treatments such as surgical resection, radiation and chemotherapy, the median survival time generally does not last over 2 years (Mercer et al., 2009). Malignant gliomas are usually not surgically curable because tumour relapses shortly after surgical removal. Even when various treatment strategies are combined, the prognosis remains dismal, due to the strong propensity of the tumour to grow into the surrounding normal brain tissues. As intracranial tumours are characterized by high infiltration and proliferation that are closely associated with morbidity and mortality, novel therapeutic approaches that inhibit primary tumour growth will greatly benefit malignant glioma patients.

The development of novel therapeutic approaches is based on better knowledge of the mechanisms that underlie the highly invasive and proliferative characteristics of malignant glioma. One critical molecule is FAK (focal adhesion kinase). FAK, a non-receptor tyrosine kinase, resides at sites of integrin clustering (the so-called ‘focal adhesions’), adapts protein–protein interaction where cells attach to the ECM (extracellular matrix) and implicates in several cellular processes such as proliferation, apoptosis, motility and invasion. FAK also transmits growth-factor-dependent signals into the cell interior (McLean et al., 2005). The synergistic signalling crosstalk between growth-factor receptors and FAK might be particularly relevant in the cancer background. Endothelial cell-specific knockout of FAK in mice impaired angiogenesis, leading to a late embryonic lethal phenotype. This observation suggests that FAK may also play an important role in vascular development (Shen et al., 2005). FAK is found at elevated levels in a wide spectrum of cancers, particularly as cancers transform into highly invasive phenotypes (Canel et al., 2006; Fujii et al., 2004; Gabarra-Niecko et al., 2003; Lark et al., 2003; Recher et al., 2004). Multiple lines of evidence have demonstrated roles for FAK in the promotion of glioma cell proliferation, survival, migration and angiogenesis. For instance, an increased expression of FAK was detected in biopsy samples of malignant glioma as compared with those of normal brain (Gutenberg et al., 2004). Moreover, elevated levels of FAK and activated FAK were found to be expressed in angiogenic blood vessels of malignant gliomas in vivo (Haskell et al., 2003). In an in vivo experiment, clones of U251 glioma cells overexpressing FAK elicited a 1.6–2.8-fold increase in tumour growth after inoculated into SCID (severe combined immunodeficiency) mouse brains (Wang et al., 2000). These findings suggest that FAK represents a promising intervention target for treatment of malignant glioma.

Various FAK-attenuating approaches have been developed, among which RNAi (RNA interference) technology shows great attracting prospects because of its ‘revolutionary’ potency and selectivity in targeted gene silencing. shRNA (small-hairpin RNA), a substitute of synthesized siRNA (small interfering RNA), has been found to achieve a longer silencing of the targeted gene and is renewable, representing an ideal tool for gene therapy, provided it is based on an appropriate vector (Sui et al., 2002). In the present study, we formulated a complex, named ‘lipoplex’, by coupling plasmids expressing FAK-targeted shRNAs to the cationic liposome DOTAP (dioleoyltrimethylammonium propane):Chol (cholesterol), which has been shown to have the potential to serve as a ‘targeted’ delivery vehicle by Ramesh and co-workers (Gopalan et al., 2004; Ito et al., 2003; Ito et al., 2004a, b; Ramesh, 2008; Ramesh et al., 2004). The lipoplex was then administered systemically to nude mice bearing xenografts derived from human malignant glioma. Tumour growth rate, tumour cell proliferation and apoptosis, MVD (microvessel density) and treatment-associated toxicity were evaluated subsequently.

2. Materials and methods

2.1. Construction of shRNA expressing plasmid

The plasmid vector expressing FAK-targeted shRNA was constructed using pGenesil-2 vector (Genesil Corp, Wuhan, China). The targeting sequence for human FAK was 5′-AACCACCTGGGCCAGTATTAT-3′ (GenBank® accession No. NM_153831), which had been proved to be specific and effective (Halder et al., 2006). KB-shRNA, which has no homology with any human and mouse gene, was constructed as control (5′-GACTTCATAAGGCGCATGC-3′). The shRNA sequence was cloned into the BamHI/HindIII restriction site of the pGenesil-2 vector. All the sequences were verified by DNA sequencing. The resulting recombinant plasmids were named FAK-shRNA and KB-shRNA respectively. Then, a large-scale preparation of plasmid DNA was extracted from DH5α Escherichia coli transformed using EndoFree Plasmid Giga Kits (Qiagen GmbH, Hilden, Germany). The concentration was determined by measuring A260/A280 ratio using UV spectrophotometry. The DNA was dissolved in sterile water and stored at −20°C before use.

2.2. Cell line and culture conditions

The human glioma cell line U251 was purchased from the A.T.C.C. and preserved by the State Key Laboratory of Biotherapy (West China Hospital of Sichuan University, Chengdu, China). The cells were cultured in DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% FBS (fetal bovine serum) and 5% penicillin/streptomycin. The cells were maintained in a humidified atmosphere of 5% CO2 at 37°C.

2.3. Transient transfection of U251 cell line

The transfection procedure was performed using Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA, U.S.A.) according to the manufacturer's recommendation. Briefly, U251 cells were seeded in six-well plate at a density of 5×105 cells/well and cultured for 24 h. Transfection was carried out when cells reached a confluence of 60−80%. Gently mixed 2 μg plasmid DNA (FAK-shRNA or KB-shRNA)/5 μl Lipofectamine™ 2000, named FAK-shRNA-Lipo or KB-shRNA-Lipo respectively, was diluted in serum-free DMEM medium to give a final volume of 200 μl, and then incubated at room temperature for 30 min. The plasmid DNA/liposome complex was added to cell cultures for 6 h of incubation, and then the medium was replaced with fresh growth medium. Serum-free growth medium was used as the control agent. After transfection, cells were cultured for another 72 h and subjected to downstream analysis.

2.4. Flow cytometry analysis and PI (propidium iodide) staining

Apoptotic cells were visualized using an Annexin V-FITC apoptosis detection kit (Nanjing Keygen Biotech. Co. Ltd), according to the manufacturer's protocol. Briefly, cells were harvested 72 h after transfection, washed twice with PBS and resuspended in 500 μl Annexin V binding. Two microlitres of FITC-conjugated Annexin V was added following an addition of 5 μl PI. After incubation for 5 min at room temperature in the dark, samples were immediately analysed using FACSCalibur™ flow cytometer (BD Biosciences, San Jose, CA, U.S.A.). Approx. 1×104 cells were collected and analysed with CELLQuest software (BD Biosciences). Morphological analysis of apoptosis was performed by PI staining. Cells in six-well plates were washed by PBS, stained with 50 mg/ml PI solutions and examined by fluorescence microscopy using Zeiss Axiovert 200 microscope. All these experiments were repeated thrice.

2.5. Synthesis of DOTAP:Chol cationic liposome and preparation of lipoplex for in vivo treatment

The DOTAP:Chol for in vivo use was prepared as described elsewhere (Templeton et al., 1997). Briefly, DOTAP and Chol were mixed at equimolar concentrations. The mixed powdered lipids were dissolved in HPLC-grade chloroform (Mallinckrodt, Chesterfield, IN, U.S.A.) to form a clear solution that was then rotated on a Buchi rotary evaporator to make a thin film. The film was dried under vacuum and rehydrated in 5% GS (glucose solution) to give a final concentration of 5 mg/ml of DOTAP:Chol. The rehydrated lipid film was then rotated in a water bath and allowed to stand overnight. After, the mixture was sonicated at a low frequency and sequentially extruded through Millipore (Millipore, Billerica, MA, U.S.A.) polycarbonate membrane of decreasing sizes (1.0, 0.45, 0.2 and 0.1 μm). The final product was stored in argon gas at 4°C. DOTAP and highly purified Chol were purchased from Avanti Polar Lipids (Avanti Polar Lipids, Alabaster, AL, U.S.A.) and Sigma-Aldrich (St. Louis, MO, U.S.A.) respectively.

Before tail vein injection, lipoplex was prepared as follows: 5 μg DNA and 25 μg DOTAP:Chol were diluted respectively in 50 μl of 5% GS to produce a 1:5 ratio of DNA (μg):liposome (μg). The DNA solution was added into the liposome solution dropwise. The mixture was gently mixed and left at room temperature for 30 min prior to injection in mice. Freshly prepared lipoplex was analysed for particle size by Malvern Zen 600 Zetasizer (Malvern Instruments, Malvern, Worcestershire, U.K.). The particle size of the lipoplex ranged between 200 and 300 nm.

2.6. In vivo tumour models and systemic treatment

The following studies were approved by the Institutional Animal Care and Treatment Committee of Sichuan University (Sichuan University, Chengdu, China). Female athymic nude mice (BALB/c, 4–6 weeks of age) were housed in standard microisolator conditions free of pathogens in accordance with institutional guidelines under approved protocol. The nude mice were inoculated subcutaneously with 5×106 U251 cells each in the right flank on day 0. Tumours were palpable 1-week later when they reached a volume of 50–60 mm3. The mice were then randomly assigned into three groups (six mice per group) to receive the following treatment: (a) 5 μg FAK-shRNA/25 μg liposome complex (FAK-shRNA lipoplex); (b) 5 μg KB-shRNA/25 μg liposome complex (KB-shRNA lipoplex); and (c) 5% GS (mock). The systemic therapy started on day 7 and was repeated three times per week (on Monday, Wednesday and Friday). Tumour diameters of each mouse were measured twice a week during the treatment period. Tumour volume of each mouse was calculated using the formula: tumour size = length×(width)2×0.52. The scheduled treatment lasted for 4 weeks. All the mice were killed after 12 times of treatment in total or when any of the mice began to appear moribund. Xenografts were excised and weighed, half of which were snap frozen immediately for Western-blot assay, and anthers were fixed in 4% (w/v) paraformaldehyde solution and embedded in paraffin for immunohistochemical analysis.

2.7. Immunohistochemical analysis

Expression of FAK, VEGF (vascular endothelial growth factor), CD34 and PCNA (proliferating-cell nuclear antigen) in xenografts was evaluated by immunohistochemical analysis carried out as described below. Rabbit anti-human FAK antibody, rabbit anti-human VEGF antibody, goat anti-mouse CD34 antibody and mouse anti-human PCNA antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.). Paraffin-embedded tumour sections were treated by standard deparaffinization and rehydrated in graded series of alcohol [100, 95, 85 and 75% (v/v) ethanol/double-distilled H2O]. Antigen retrieval was performed by heating slides in an autoclave at 120°C for 3 min after pressure gaining in 10 mM citrate buffer solution (pH 6.0). Endogenous peroxidase was inactivated by incubating slides in 3% H2O2 solution for 15 min. Non-specific antibody binding was blocked with normal goat (for FAK, VEGF and PCNA) or rabbit (for CD34) serum for 30 min at 37°C before incubation overnight at 4°C with primary antibodies. After being rinsed in PBS, sections of tissue were incubated with goat anti-rabbit antibody for FAK and VEGF, rabbit anti-goat antibody for CD34 and goat anti-mouse antibody for PCNA. After 40 min incubation with peroxidase-labelled SAB (streptavidin–biotin) reagents at 37°C, immunostaining was performed using DAB (diaminobenzidine) peroxide solution. Slides were observed under microscopy to end the reaction timely. Cell nuclei were counterstained with haematoxylin. To quantify PCNA expression, the number of positive cells was counted and the ratio of immunoreactive cells to the total number of cells counted in five random fields (×400) is calculated. To quantify MVD, the mean of the absolute number of the microvessels per high-power field of five random fields (×400) is regarded as the index for MVD.

2.8. Western-blot analysis

Tumour tissues of each group were homogenized and lysed in RIPA lysis buffer containing 50 mM Tris/HCl (pH 7.4), 0.25% sodium deoxycholate, 150 mM NaCl, 1% Nonidet P40, 1 mM EDTA, 1 mM NaF, 1 mM Na3V4 and 1 mM cocktail (Sigma). Protein concentrations were determined on diluted samples using Bio-Rad protein assay (Bio-Rad, Hercules, CA, U.S.A.). Equal amounts of protein (15–50 μg) were loaded on to SDS/10% PAGE gel by electrophoresis and transferred on to PVDF [poly(vinylidene difluoride)] membranes (Millipore, Bedford, MA, U.S.A.). The membranes were rinsed in TBS with 0.1% Tween-20 and blocked for 1 h at room temperature with 50 g/l non- fat dried skimmed milk powder in TBS. Proteins of FAK and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) were immunoblotted with rabbit anti-human FAK antibody and mouse anti-human GAPDH antibody (Santa Cruz Biotechnology). The blots were labelled with HRP (horseradish peroxidase)-conjugated secondary antibody respectively, goat anti-rabbit IgG for FAK and goat anti-mouse IgG, then detected by chemiluminescence. The bands (TIFF imaged) were analysed digitally and quantified using Quantity One 4.5.2 (Bio-Rad).

2.8.1. In situ TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) assay

Tissue DNA fragmentation morphology was detected by TUNEL staining using DeadEnd™ Fluorometric TUNEL System (Promega, Madison, WI, U.S.A.) according to the manufacturer's protocol. Briefly, paraffin-embedded sections were deparaffinized and rehydrated, then pretreated with 20 μg/ml proteinase K for 10 min. Cells on slides were incubated with rTdT incubation buffer at 37°C for 1 h to allow the tailing reaction to occur. Cell nuclei with dark-green fluorescent staining were defined as TUNEL-positive nuclei. TUNEL-positive cells were visualized and analysed under a fluorescence microscope (Olympus, Tokyo, Japan). To quantify TUNEL-positive cells, the number of green-fluorescence-positive cells was counted in ten random 0.011 mm2 fields at ×200 magnification.

2.9. Toxicity assessment

Toxicity indexes such as weight loss, ruffled fur, diarrhoea, anorexia, cachexia or toxic deaths were continuously observed until the time of killing and recorded. Samples of organs (heart, liver, spleen, kidney, lung, etc) were fixed in 4% paraformaldehyde solution, embedded in paraffin. Sections of 4 μm were stained with H&E (haematoxylin & eosin) and observed for possible toxic effects by two separate pathologists.

2.10. Statistical analysis

Statistical analysis was performed using Mann–Whitney U test for the analysis of variants. P<0.05 was considered statistically significant. The Statistical Package for the Social Sciences (SPSS, Inc., Chicago, IL, U.S.A.) was applied for all statistical analyses.

3. Results

3.1. FAK knockdown induces apoptosis in vitro

To determine the effect of FAK knockdown on tumour cells in vitro, we performed flow cytometry for quantitative evaluation of apoptosis. U251 cells were harvested for flow cytometry analysis 72 h after being transfected with plasmid expressing FAK shRNA or KB shRNA. As shown in Figure 1(A), the FAK-shRNA-Lipo group has a distinctly higher amount of apoptotic cells than the KB-shRNA-Lipo group and the blank control group, with 46.7, 24.0 and 7.0% apoptotic cells respectively. Next we stained the cells in six-well plate with PI solution 72 h after transfection and observed morphological changes under a fluorescence microscope. Consistent with the results of quantitative analysis, cells in the FAK-shRNA-Lipo group exhibited morphological changes typical for apoptosis. As arrows in Figure 1(Bc) indicate, most of the cells were detached from the well surface with the remaining ones displaying changes such as brightly red-fluorescent nuclei, condensation of nuclear chromatin, nuclear fragmentation and apoptotic bodies. In contrast, as shown in Figures 1(Ba) and 1(Bb), cells in the other groups anchor themselves to the well surface, showing no obvious changes characteristic of apoptosis.

3.2. FAK knockdown inhibits tumour xenograft growth in vivo

The in vitro data presented above demonstrated that suppression of FAK has an inhibitory effect on cultured tumour cells, and then we asked whether suppression of FAK also inhibits tumour cells growth in vivo. To facilitate the systemic delivery of plasmids, we formulated a complex, namely ‘lipoplex’, by enclosing plasmids (5 μg) expressing specific shRNAs with the cationic liposome DOTAP:Chol (25 μg). The lipoplex was injected repeatedly via the tail vein into nude mice bearing tumours derived from the U251 glioma cell line. During the course of treatment, we observed that tumour growth was delayed significantly (P<0.05) from day 21 after inoculation until the day of killing. Treatment with FAK-shRNA lipoplex resulted in 70.9 and 69.7% reduction in tumour volume compared with the blank control and negative control respectively (Figure 2A). These results show that intravenous administration of low-dose therapeutic plasmids expressing FAK-shRNA in a repeated manner has a lasting inhibitory effect on tumour development. When the mice were killed, the xenograft tumours were excised and weighed. Consistently, treatment with FAK-shRNA lipoplex resulted in 66.0 and 61.2% reduction in tumour weight compared with the blank control and negative control respectively (Figure 2B).

3.3. FAK-shRNA knockdown the FAK expression in vivo

To prove the correlation between knockdown of FAK expression and therapeutic effects observed, and confirm the specificity and potency of FAK-targeted shRNA, we examined the level of FAK protein in harvested tumours by Western-blot analysis and immunohistochemical staining. As shown in Figure 3(A), we found that FAK-shRNA lipoplex yielded 87.35% knockdown of FAK expression, while KB-shRNA lipoplex and 5% GS had almost no detectable effect on FAK expression. Next, the gross distribution of FAK immunopositive cells in the tumour tissues was evaluated and the results indicated that tumours from the mice treated with FAK-shRNA lipoplex showed an overall decrease of FAK staining, while tumours belonging to the mice receiving KB-shRNA lipoplex and 5% GS exhibited significantly more FAK staining, as shown in Figure 3(B). The result of immunohistochemistry for FAK was in agreement with that of the Western-blot analysis. Thus, we confirmed the specificity and potency of FAK-targeted shRNA and demonstrated that it was the knockdown of FAK protein that inhibited the tumour growth in vivo.

3.4. FAK knockdown inhibited cell proliferation and induced apoptosis in vivo

We have demonstrated that FAK-targeted therapy slowed tumour growth rate in vivo. To explore the underlying mechanisms, we first performed PCNA staining to determine the effect of FAK knockdown on tumour cell proliferation. As shown in Figure 4(A), the positive rates of PCNA were 23.6±1.5, 58.3±2.1 and 76.2±1.4% in tumours belonging to the mice treated with FAK-shRNA lipoplex, KB-shRNA lipoplex and 5% GS respectively. An apparent reduction of PCNA expression was observed in tumours from the mice receiving FAK-shRNA lipoplex, compared with controls (P<0.05). We next performed the in situ TUNEL assay to determine the effect of FAK knockdown on tumour cell apoptosis. As shown in Figure 4(B), the apoptosis index of the FAK-shRNA lipoplex group (33.6±4.8%) was significantly higher than that of the KB-shRNA lipoplex group (3.8±0.5%) and the 5% GS group (2.3±0.6) (P<0.05).

3.5. FAK knockdown inhibited angiogenesis in vivo

Emerging evidence has unfolded a role of FAK in promotion of angiogenesis in malignant gliomas (Haskell et al., 2003). We then inquired into the MVD in the tumours harvested from each group. As shown in Figure 5(A), the most significant reduction in the number of newly formed vessels occurred in tumours from the mice treated with FAK-shRNA lipoplex (12.6±1.3%), compared with the KB-shRNA lipoplex and 5% GS groups, with 46.1±2.4 and 57.2±3.6% respectively (P<0.05). Based on the recent evidence that inhibited FAK activity associates with down-regulation of angiogenic factors (Mitra et al., 2005), we further examined the expression of the critical angiogenic factor, VEGF, in the harvested tumours. As shown in Figure 5(B), tumours derived from the FAK-shRNA lipoplex group exhibited much less staining for VEGF than that in the KB-shRNA lipoplex and 5% GS groups. In addition, it is noteworthy that there were no significant differences between the KB-shRNA lipoplex and 5% GS groups (P>0.05) in FAK expression, the percentage of PCNA- or TUNEL- positive cells or MVD, indicating that the negative control vector has little antitumour effect.

3.6. Assessment of potential toxicity

To evaluate treatment-related toxicity, we observed and recorded health status indexes, including weight loss, ruffled fur, diarrhoea, anorexia, cachexia or toxic deaths through the whole experiment. No mentioned signs above were found in any group. Furthermore, no overt pathologic changes were found in sections of organs (lungs, livers, kidneys, hearts, kidneys, spleens, brains etc.) of any group by microscopic observation.

4. Discussion

In the present study, we delivered plasmids expressing FAK-targeted shRNA systemically in a xenograft model of human malignant glioma in vivo. We found that knockdown of FAK had pronounced antitumour effects on malignant glioma, resulting in approx. 70% reduction in tumour volume compared with controls. Thus, we described a successful therapeutic intervention in the established tumour model with FAK serving as an intervention target.

To uncover potential mechanisms behind the pronounced antitumour efficacy, we analysed the molecular changes occurring with a concomitant inhibition of FAK in vivo. Consistent with established evidence linking FAK signalling to apoptosis (Sonoda et al., 2000), significantly increased apoptosis was observed in FAK-deficient tumours compared with controls. Decreased proliferation was also observed, probably related to decreased Ras activity (Hecker et al., 2004). Hecker et al. have proved that overexpression of FAK in U-251MG cells in aggregate suspension culture reduced the amount of p120RasGAP complexed with active Ras, therefore facilitating Ras activity. It has been shown that transgenic mice expressing constitutively active Ras under the control of an astrocyte-specific promoter exhibit astrocyte proliferation in vivo ( Guha et al., 1997; Ding et al., 2001). In addition, Ras activity has been reported to be elevated in malignant astrocytoma biopsy samples in the absence of Ras gain of function mutations (Guha, 1998). Moreover, MVD was also significantly decreased in FAK-deficient tumours that were found to exhibit less VEGF staining in our study. Therefore, although FAK knockdown is primarily the direct antitumour approach, it should also be regarded as anti-angiogenic, at least in malignant glioma. The anti-angiogenic effect together with enhanced apoptosis contributes to the antitumour effect. The exact mechanism for the anti-angiogenic effect mediated by FAK inhibition remains unknown; however, emerging evidence has suggested several pathways as follows: (i) cell contact-mediated induction of VEGF transcription acts in an FAK-dependent way (Sheta et al., 2000), and thus FAK inhibition may block the induction of VEGF through this pathway. (ii) An elevated expression of FAK has been detected in angiogenic blood vessels of malignant glioma (Haskell et al., 2003). Given the facts that angiogenic endothelial cells in tumours preferentially bind and internalize the cationic liposome DOTAP:Chol that we used in the present study (Thurston et al., 1998), we speculate that FAK-targeted shRNA transferred into angiogenic blood vessels may inhibit proliferation, survival and migration of angiogenic endothelial cells, resulting in impaired cell sprouting and tube formation. However, further studies are required to elucidate the issues mentioned above.

Supporting our findings, recent studies have shown encouraging outcomes using different delivery vehicles and tumour models. In one study, polyethylenimine-complexed plasmid expressing FAK-shRNA suppressed primary melanoma tumour growth and metastasis (Li et al., 2007). In another study, intratumoural delivery of FAK-shRNA with cationic liposome inhibited the growth of prostate and breast cancers (Tsutsumi et al., 2009). There is also experimental evidence that neutral liposome-complexed FAK siRNA functioned as ovarian carcinoma therapeutics (Halder et al., 2006). These findings suggest FAK as an attractive therapeutic target and RNAi as a promising therapeutic approach for cancer therapy.

Potent sequence-specific gene silencing by RNAi therapeutics promises the ultimate level of specificity at the cellular level. However, efficacy of an RNAi-based cancer therapeutic is largely dependent on the method of delivery. An ideal delivery vehicle must be able to selectively target tumours compared with normal tissue and smoothly enter tumour cells following systemic administration. The vehicle we choose is the cationic liposome DOTAP:Chol. Recently, our colleagues have successes in silencing of the PI3K (phosphoinositide 3-kinase)-110α gene in an ovarian cancer model using DOTAP:Chol (Zhang et al., 2009). Previous studies focused on DOTAP:Chol have revealed several important facts about its features. One is that DOTAP:Chol or DOTAP:Chol–DNA complexes are preferentially taken up by angiogenic endothelial cells rather than normal endothelial cells, whereas anionic, neutral or sterically stabilized neutral liposomes are not (Thurston et al., 1998). Most importantly, unlike some other cationic liposomes that are not tissue specific and tend to accumulate in lung and liver, DOTAP:Chol exhibits a much higher affinity for tumour sites than for other tissues (Ito et al., 2003). Moreover, we selected a dose of 5 μg to treat nude mice in the in vivo experiment. There are several reasons for the low dose. On the one hand, it is known that when integrated in the host genome, shRNA can be continuously synthesized in host cells and provide durable gene knockdown effect. On the other hand, toxicity must be considered. It has been reported that complex formation with plasmid DNA increases the cytotoxicity of cationic liposome in a dose-dependent manner (Nguyen et al., 2007). In the present study, chronic intravenous administration of plasmid DNA was followed by delayed tumour growth despite the limited dosage. In the absence of pathological changes identified in lungs or livers or any other organs of the mice, this regimen was well tolerated. Additionally, further studies should be taken into consideration. Although the biodistribution and tumour accumulation of plasmid–liposome complex were investigated intensively (Mahato et al., 1998; Zhang et al., 2008), the detailed biodistribution of systematic administration of FAK-shRNA lipoplex need to be further investigated.

In summary, we have demonstrated that treatment with DOTAP:Chol complexed plasmid expressing FAK-shRNA was highly effective in inhibiting malignant glioma growth by both direct and indirect antitumour mechanisms. Our work suggests that this approach could be a potential therapeutic approach against malignant glioma.

Author contribution

Guan-Jie Wang and Yong-Ping Ma carried out study design, literature research, experimental studies, data acquisition, data analysis, statistical analysis and manuscript preparation. Yang Yang participated in the design of the study and drafted the manuscript. Na Zhang and Wei Wang carried out the molecular genetic studies, participated in the sequence alignment. Sheng-Yong Liu participated in the animal study. Li-Juan Chen prepared the cationic lipids. Yu Jiang participated in the immunoassays. Xia Zhao and Yu-Quan Wei participated in literature research, data analysis and manuscript editing. Hong-Xin Deng conceived the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


This work was supported by The National Key Basic Research Program (973 Program) of China [grant number 2010CB529900] and Hi-tech Research and Development Program (863 Program) of China [grant numbers 2007AA021008 and 2009ZX09102-241].


Canel, M, Secades, P, Rodrigo, JP, Cabanillas, R, Herrero, A and Suarez, C (2006) Overexpression of focal adhesion kinase in head and neck squamous cell carcinoma is independent of fak gene copy number. Clin Cancer Res 12, 3272-9
Crossref   Medline   1st Citation  

Ding, H, Roncari, L, Shannon, P, Wu, X, Lau, N and Karaskova, J (2001) Astrocyte-specific expression of activated p21-ras results in malignant astrocytoma formation in a transgenic mouse model of human gliomas. Cancer Res 61, 3826-36
Medline   1st Citation  

Fujii, T, Koshikawa, K, Nomoto, S, Okochi, O, Kaneko, T and Inoue, S (2004) Focal adhesion kinase is overexpressed in hepatocellular carcinoma and can be served as an independent prognostic factor. J Hepatol 41, 104-11
Crossref   Medline   1st Citation  

Gabarra-Niecko, V, Schaller, MD and Dunty, JM (2003) FAK regulates biological processes important for the pathogenesis of cancer. Cancer Metastasis Rev 22, 359-74
Crossref   Medline   1st Citation  

Gopalan, B, Ito, I, Branch, CD, Stephens, C, Roth, JA and Ramesh, R (2004) Nanoparticle based systemic gene therapy for lung cancer: molecular mechanisms and strategies to suppress nanoparticle-mediated inflammatory response. Technol Cancer Res Treat 3, 647-57
Medline   1st Citation  

Guha, A (1998) Ras activation in astrocytomas and neurofibromas. Can J Neurol Sci 25, 267-81
Medline   1st Citation  

Guha, A, Feldkamp, MM, Lau, N, Boss, G and Pawson, A (1997) Proliferation of human malignant astrocytomas is dependent on Ras activation. Oncogene 15, 2755-65
Crossref   Medline   1st Citation  

Gutenberg, A, Bruck, W, Buchfelder, M and Ludwig, HC (2004) Expression of tyrosine kinases FAK and Pyk2 in 331 human astrocytomas. Acta Neuropathol 108, 224-30
Medline   1st Citation  

Halder, J, Kamat, A, Landen, CN Jr, Han, LY, Lutgendorf, SK and Lin, YG (2006) Focal adhesion kinase targeting using in vivo short interfering RNA delivery in neutral liposomes for ovarian carcinoma therapy. Clin Cancer Res 12, 4916-24
Crossref   Medline   1st Citation   2nd  

Haskell, H, Natarajan, M, Hecker, TP, Ding, Q, Stewart, J Jr and Grammer, JR (2003) Focal adhesion kinase is expressed in the angiogenic blood vessels of malignant astrocytic tumors in vivo and promotes capillary tube formation of brain microvascular endothelial cells. Clin Cancer Res 9, 2157-65
Medline   1st Citation   2nd   3rd  

Hecker, TP, Ding, Q, Rege, TA, Hanks, SK and Gladson, CL (2004) Overexpression of FAK promotes Ras activity through the formation of a FAK/p120RasGAP complex in malignant astrocytoma cells. Oncogene 23, 3962-71
Crossref   Medline   1st Citation  

Ito, I, Began, G, Mohiuddin, I, Saeki, T, Saito, Y and Branch, CD (2003) Increased uptake of liposomal-DNA complexes by lung metastases following intravenous administration. Mol Ther 7, 409-18
Crossref   Medline   1st Citation   2nd  

Ito, I, Ji, L, Tanaka, F, Saito, Y, Gopalan, B and Branch, CD (2004a) Liposomal vector mediated delivery of the 3p FUS1 gene demonstrates potent antitumor activity against human lung cancer in vivo. Cancer Gene Ther 11, 733-9
Crossref   Medline   1st Citation  

Ito, I, Saeki, T, Mohuiddin, I, Saito, Y, Branch, CD and Vaporciyan, A (2004b) Persistent transgene expression following intravenous administration of a liposomal complex: role of interleukin-10-mediated immune suppression. Mol Ther 9, 318-27
Medline   1st Citation  

Lark, AL, Livasy, CA, Calvo, B, Caskey, L, Moore, DT and Yang, X (2003) Overexpression of focal adhesion kinase in primary colorectal carcinomas and colorectal liver metastases: immunohistochemistry and real-time PCR analyses. Clin Cancer Res 9, 215-22
Medline   1st Citation  

Li, S, Dong, W, Zong, Y, Yin, W, Jin, G and Hu, Q (2007) Polyethylenimine-complexed plasmid particles targeting focal adhesion kinase function as melanoma tumor therapeutics. Mol Ther 15, 515-23
Crossref   Medline   1st Citation  

Mahato, RI, Anwer, K, Tagliaferri, F, Meaney, C, Leonard, P and Wadhwa, MS (1998) Biodistribution and gene expression of lipid/plasmid complexes after systemic administration. Hum Gene Ther 9, 2083-99
Crossref   Medline   1st Citation  

McLean, GW, Carragher, NO, Avizienyte, E, Evans, J, Brunton, VG and Frame, MC (2005) The role of focal-adhesion kinase in cancer – a new therapeutic opportunity. Nat Rev Cancer 5, 505-15
Crossref   Medline   1st Citation  

Mercer, RW, Tyler, MA, Ulasov, IV and Lesniak, MS (2009) Targeted therapies for malignant glioma: progress and potential. BioDrugs 23, 25-35
Crossref   Medline   1st Citation  

Mitra, S, Molina, J, Hanson, D, Hsia, D, Mikolon, D and Chi, A (2005) Focal adhesion kinase (FAK) activity promotes tumor growth and neo-vascularization via up-regulation of angiogenic factors. AACR Meeting Abstracts 2005, 475-a
1st Citation  

Nguyen, LT, Atobe, K, Barichello, JM, Ishida, T and Kiwada, H (2007) Complex formation with plasmid DNA increases the cytotoxicity of cationic liposomes. Biol Pharm Bull 30, 751-7
Crossref   Medline   1st Citation  

Ramesh, R (2008) Nanoparticle-mediated gene delivery to the lung. Methods Mol Biol 433, 301-31
Medline   1st Citation  

Ramesh, R, Ito, I, Saito, Y, Wu, Z, Mhashikar, AM and Wilson, DR (2004) Local and systemic inhibition of lung tumor growth after nanoparticle-mediated mda-7/IL-24 gene delivery. DNA Cell Biol 23, 850-7
Medline   1st Citation  

Recher, C, Ysebaert, L, Beyne-Rauzy, O, Mansat-De Mas, V, Ruidavets, JB and Cariven, P (2004) Expression of focal adhesion kinase in acute myeloid leukemia is associated with enhanced blast migration, increased cellularity, and poor prognosis. Cancer Res 64, 3191-7
Crossref   Medline   1st Citation  

Shen, TL, Park, AY, Alcaraz, A, Peng, X, Jang, I and Koni, P (2005) Conditional knockout of focal adhesion kinase in endothelial cells reveals its role in angiogenesis and vascular development in late embryogenesis. J Cell Biol 169, 941-52
Crossref   Medline   1st Citation  

Sheta, EA, Harding, MA, Conaway, MR and Theodorescu, D (2000) Focal adhesion kinase, Rap1, and transcriptional induction of vascular endothelial growth factor. J Natl Cancer Inst 92, 1065-73
Crossref   Medline   1st Citation  

Sonoda, Y, Matsumoto, Y, Funakoshi, M, Yamamoto, D, Hanks, SK and Kasahara, T (2000) Anti-apoptotic role of focal adhesion kinase (FAK). Induction of inhibitor-of-apoptosis proteins and apoptosis suppression by the overexpression of FAK in a human leukemic cell line, HL-60. J Biol Chem 275, 16309-15
Crossref   Medline   1st Citation  

Sui, G, Soohoo, C, Affarel, B, Gay, F, Shi, Y and Forrester, WC (2002) A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc Natl Acad Sci USA 99, 5515-20
Crossref   Medline   1st Citation  

Templeton, NS, Lasic, DD, Frederik, PM, Strey, HH, Roberts, DD and Pavlakis, GN (1997) Improved DNA: liposome complexes for increased systemic delivery and gene expression. Nat Biotechnol 15, 647-52
Crossref   Medline   1st Citation  

Thurston, G, McLean, JW, Rizen, M, Baluk, P, Haskell, A and Murphy, TJ (1998) Cationic liposomes target angiogenic endothelial cells in tumors and chronic inflammation in mice. J Clin Invest 101, 1401-13
Crossref   Medline   1st Citation   2nd  

Tsutsumi, K, Yamaura, T, Nakajima, M, Honda, T and Kasaoka, T (2009) Silencing of focal adhesion kinase by tumor direct injection of small interfering RNA decreases in vivo tumor growth. Cancer Biol Ther 8, 1292-9
Crossref   Medline   1st Citation  

Wang, D, Grammer, JR, Cobbs, CS, Stewart, JE Jr, Liu, Z and Rhoden, R (2000) p125 focal adhesion kinase promotes malignant astrocytoma cell proliferation in vivo. J Cell Sci 113, 4221-30
Medline   1st Citation  

Zhang, X, Deng, HX, Zhao, X, Su, D, Chen, XC and Chen, LJ (2009) RNA interference-mediated silencing of the phosphatidylinositol 3-kinase catalytic subunit attenuates growth of human ovarian cancer cells in vitro and in vivo. Oncology 77, 22-32
Crossref   Medline   1st Citation  

Zhang, Y, Bradshaw-Pierce, EL, Delille, A, Gustafson, DL and Anchordoquy, TJ (2008) In vivo comparative study of lipid/DNA complexes with different in vitro serum stability: effects on biodistribution and tumor accumulation. J Pharm Sci 97, 237-50
Crossref   Medline   1st Citation  

Received 15 April 2010/1 December 2010; accepted 20 January 2011

Published as Cell Biology International Immediate Publication 20 January 2011, doi:10.1042/CBI20100243

© The Author(s) Journal compilation © 2011 Portland Press Limited

ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)