Springer 2006

Journal of Neuro-Oncology (2006) 76: 1–11 DOI 10.1007/s11060-005-3029-3

Laboratory Investigation

The effects of antisense AKT2 RNA on the inhibition of malignant glioma cell growth in vitro and in vivo Peiyu Pu1, Chunsheng Kang1, Jie Li1, Hao Jiang2 and Jinquan Cheng3 1 Department of Neurosurgery, Tianjin Medical University General Hospital, Laboratory of Neuro-Oncology, Tianjin Neurological Institute, Tianjin, People’s Republic of China; 2William T. Gossett Neurology Laboratories, Department of Neurology, Henry Ford Health System, Detroit, MI, USA; 3Department of Pathology, H Lee Moffitt Cancer Center, University of South Florida College of Medicine, Tampa, FL, USA

Key words: antisense AKT2, apoptosis, cell proliferation, malignant gliomas Summary The oncogenic role of AKT2 in the development of malignant gliomas was examined by using antisense approach. AKT2 expression was significantly inhibited in rat C6 glioma cells transfected with antisense AKT2 cDNA construct (LXSN–AS–AKT2). In addition, the transfected cells proliferated at a lowered level and apoptosis was induced. For in vivo studies, parental C6 cells and C6 cells transfected with LXSN–AS–AKT2 were implanted stereotactically into the right caudate nucleus of SD rats (control C6 group and transfected group). The rats bearing well-established C6 gliomas were treated with LXSN–AS–AKT2 DNA or LXSN (empty vector)-lipofectamine complexes intratumorally (treated group and control treated group). The mean survival of the rats of control C6 group and treated control group was 17.8±0.92 days and 17.5±1.10 days, respectively. The mean survival of the rats of transfected and treated group was significantly prolonged. MR images revealed distinct cerebral tumor foci in all of the control rats, whereas four rats in transfected group did not develop tumors and the tumor foci in five rats of treated group were regressed and disappeared. The expression of AKT2, PCNA, MMP2/9, and cyclin D were inhibited in the tumors of rats in transfected and treated groups while GFAP expression was increased. These results suggest that AKT pathway may play an important role in the development and progression of gliomas. AntiAKT approach will open a new perspective for a targeted molecular therapy of malignant gliomas.

Introduction Considerable evidences have demonstrated that a number of growth factors and their receptors, such as bFGF, PDGF, IGF, VEGF, EGFR, are involved in the tumorigenesis of gliomas [1–10]. PI3K/AKT/protein kinase B (PKB) is one of the important downstream signaling pathways involved in cell survival and plays a central role in tumorigenesis [11]. AKT is a subfamily of the serine/threonine kinases that includes AKT1, AKT2, and AKT3. All three AKT isoforms are composed of an N-terminal pleckstrin homology (PH) domain, a central kinase catalytic domain and a C-terminal hydrophobic regulatory domain. Activation of AKT is PI3K-dependent. Growth factors and their receptors have been shown to activate PI3K, which in turn phosphorylates phosphotidylinositol-3,4-biphosphate (PIP2) to phosphotidylinositol-3,4,5-triphosphate (PIP3). PIP3 binds to PH domain of AKT resulting in the phosphorylation on two key residues: Thr308 and Ser473, both are required for AKT activation. Several downstream targets of AKT that contribute to the cell proliferation and survival have been identified. AKT exerts its antiapoptotic effects by phosphorylating and inactivating the pro-apoptotic BAD, procaspase-9, and forkhead transcription factors which are known to induce expression of Fas ligand. Moreover, AKT induces

NFjB-dependent transcription of pro-survival genes including BCL-xL, C-Myb, etc. AKT dysregulates cell cycle progression by inhibiting p27 and p21 and inactivates GSK3, resulting in the accumulation of b-catenin and the increase of transcription of cyclin D [11–18]. AKT promotes nuclear entry of mdm2, leading to degradation of p53 that may also be involved in uncontrolled cell proliferation [19]. In addition, AKT enhances telomerase activity by phosphorylation of hTERT and enables the cells to have unlimited replicative potential [20]. AKT promotes cell invasiveness and angiogenesis by stimulating secretion of MMPs [21–23] and activating endothelial nitric oxide synthase (eNOS) [24]. Because of its pleiotropic effects, AKT has been considered to play a central role in tumorigenesis and can be used as a potential therapeutic target [11,15]. Among three isoforms of AKT, AKT2 has been shown to be primarily involved in human cancer. It has been reported that AKT2 is amplified and/or overexpressed in a variety of human cancers, such as ovarian, breast, pancreatic, prostate, thyroid cancers and squamous cell carcinoma of oral cavity etc [25–33]. However, there are only a few reports on the elevated expression of AKT in human gliomas [34–37]. Our previous study showed that the expression of AKT2 and phosphorylation of AKT is greatly enhanced in 50 human gliomas with different grades as compared to 6 normal human

2 brain tissues and the increase is correlated positively with the tumor grade (unpublished data). In the current study, we intend to examine whether overexpression of AKT2 plays a pivotal role in the tumorigenesis of gliomas. Rat C6 glioma cells containing high levels of endogenous AKT2 were transfected with an antisense AKT2 construct and alterations of malignant phenotype and tumorigenicity were examined in vitro and in vivo using cell culture and animal model, respectively.

Material and methods Plasmid constructs Plasmid carrying antisense AKT2 or LXSN–AS–AKT2 was constructed by ligating a 1.2-kb fragment from a human AKT2 cDNA clone into the LXSN retroviral vector in the antisense orientation and the fragment was generated by deleting 70 amino acids from the C-terminus of the open reading frame of AKT2. Plasmid carrying activated AKT2 or myristoylated AKT2 (MyrAKT2) was constructed by inserting a fragment of myristoylation signal of c-Src at the amino-terminal site of AKT2 cDNA and enables it in binding to the cell membrane and activating itself.

Growth rate examination The MTT assay was performed to measure the cell proliferation. Transfected and control C6 cells (4103 cells) were plated into each well of a 96-well plate in triplicate. On each day of consecutive 6 days, 20 ll MTT (5 mg/ml) was added to each well, and the cells were incubated at 37 C for additional 4 h, then the reaction was stopped by lysing the cell with 200 ll of DMSO for 5 min and quantification measurements (optical density) were obtained at the wavelength of 570 nm, and expressed as a percentage of control. Cell proliferation was also evaluated by PCNA immunostaining using ABC-peroxidase method. Briefly, cultured cells on coverslips were fixed with acetone and incubated with primary PCNA antibody (1:100 dilution, Santa Cruz, USA) overnight at 4 C, then incubated with a biotinylated secondary antibody (1:200 dilution) at room temperature for 1 h, followed by the incubation with ABC-peroxidase reagent (1:200 dilution, Vector, USA) for an additional 1 h, washed with PBS and stained with 3,3¢ diaminobenzidine (30 mg dissolved in 100 ml of Tris buffer containing 0.03% H2O2) for 5 min, rinsed in water and counterstained with hematoxylin. The percentage of the positive staining cells in a total number of 500–1000 cells was determined under 400 magnification by a light microscope.

Cell culture and transfection Rat C6 glioma cells were grown in Dulbecco’s-modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS). Cells (1105) were plated in 60-mm cell culture dishes and grown overnight at 37 C with 5% CO2 and 95% air until they were 50–80% confluent. The rat C6 glioma cells were transfected with LXSN– AS–AKT2 construct and empty vector LXSN using lipofectamine (Invitrogen, USA). Stable transfectants were selected using G418 as previously reported [38]. Three LXSN–AS–AKT2 transfected clones were selected and expanded for further studies. Western blot analysis Total protein lysates (40 lg/sample), prepared from parental C6 glioma cells and the cells transfected with plasmids described above, were separated by SDSPAGE. The separate proteins were transferred to PVDF membranes. The blot was incubated with primary antibody against AKT2 or GFAP (1:500 dilution), followed by incubation with HRP-conjugated secondary antibody (1:1000 dilution). The specific protein was detected using a Super signal protein detection kit (Pierce, USA). After washing with stripping buffer, the PVDF membrane was reprobed with antibody against b-actin (1:500 dilution) using the same procedures described above. In situ hybridization Using AKT2 cDNA as probe, in situ hybridization was performed with DIG DNA labeling kit and Digoxenium nucleic acid detection kit (Boehringer Mannheim, Germany) according to the manufacturer’s procedure.

Flow cytometry analysis Transfected and control cells in a log phase of growth were harvested by trypsinization. Cells were incubated with RNase at 37 C for 30 min. Nuclei of cells were stained with propidium iodide for additional 30 min. Total of 10,000 nuclei were examined in a FACS Caliber flow cytometer (Becton Dickinson, USA) and DNA histograms were analyzed by Modifit software. Detection of apoptosis Apoptosis was detected by TUNEL method using in situ cell death kit (Boehringer Mannheim, Germany) according to supplier’s instruction. Apoptotic index was calculated as the number of apoptotic cells per 500–1000 total cells counted under the 400 magnification by a light microscope. Tumor growth in vivo Adult male Sprague-Dawley rats weighing between 200 and 250 g were divided randomly into four groups (10 rats per group): (1) Control C6 group: The rats were anesthetized by an intraperitoneal injection of 10% chloral hydrate (300 mg/kg), then fixed in a stereotactic apparatus. A 1.2-mm burr hole was drilled in the right side of the skull 1 mm anterior and 3.0–3.5 mm lateral to bregma to expose the dura. Using a microliter syringe equipped with a 26-guage needle and connected to the manipulating arm of the stereotactic apparatus, 1106 of parental C6 glioma cells in 10 ll serum-free DMEM were injected into the caudate nucleus at a

3 depth of 4.0–4.5 mm from the dura over a 10 min period. The needle was left in place for 10 min and then slowly withdrawn. The burr hole was filled with bone wax and the scalp wound was closed with silk threads; (2) Transfected group: The same procedures described above were performed in the rats. However, C6 glioma cells transfected with AS–AKT2 RNA were injected, instead of parental C6 cells; (3) Treated group: C6 glioma cells were injected into the right caudate nucleus, as was the case with control group. As the tumor formation demonstrated in MRI on day 5 following injection, 4 lg of plasmid LXSN–AS– AKT2 DNA in 2 ll of DMEM mixed with 13 ll of lipofectamine was injected into the tumor site by using the same coordinates with stereotactic guidance on day 5 and 7; (4) Treated control group: Instead of using plasmid LXSN–AS–AKT2 DNA, the empty vector LXSN DNA–lipofectamine complexes were injected into the tumor site in the same manner as treated group, The general behavior and survival of the rats in each group were observed. The enhanced MRI with Gd-DTPA (gadolium-diethylenetriamine pentaacetic acid) was used for monitoring the tumor size at different periods in the same animal and comparison of the development of tumors in different group of rats. The performance of MRI has been described previously [38]. Whenever the rats died naturally or were sacrificed at one of various time points, their brains were removed. The gross morphological characteristics and histopathological changes were examined as compared to the MRI features of the tumors. The expression of AKT2, PCNA, MMP2/9 and cyclin D1 were examined on paraffin-embedded sections of the homograft tumor tissues by immunohistochemical staining with ABC-peroxidase method. All the antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, USA).

Figure 1. Western blot analysis of AKT2 expression in C6 cells transfected with AS–AKT2 constructs. C6: Parental C6 cells; LXSN: Cells transfected with empty vector; AS1–3; Three C6 cell clones transfected with AS–AKT2 constructs.

Results AKT2 expression in C6 glioma cells transfected with AS– AKT2 RNA As shown by Western blot analysis and in situ hybridization, the AKT2 protein and mRNA were greatly decreased in three C6 clones stably transfected with AS– AKT2 (Figures 1 and 2), indicating that the AKT2 expression was significantly inhibited by AS–AKT2 RNA in C6 glioma cells. Proliferation of transfected C6 glioma cells The AS–AKT2-transfected C6 cells proliferated at a significantly lower level than the parental C6 cells and the cells transfected with empty LXSN vector as measured by MTT assay (Table 1). Similarly, the PCNA positive staining cells were also markedly decreased than that of control cells (Table 2). The flow cytometric analysis showed that the S phase fraction (SPF) was lowered in C6 cells transfected with AS–AKT2 RNA (Figure 3, Table 3), suggesting the tendency to delay the cell cycle progression in these cells.

Statistical analysis

GFAP expression of transfected C6 cells

The data obtained in this study were analyzed by oneway ANOVA test and LSD using SPSS 10.0 version software.

As shown by Western blot analysis, GFAP expression level was extremely lowered or barely visible in control C6 cells, while its expression was significantly

Figure 2. In situ hybridization of cells transfected with AS–AKT2 constructs. AKT2 mRNA expression is significantly downregulated in cells transfected with AS–AKT2 construct (200).

4 Table 1. Proliferation rate of C6 cells transfected with antisense AKT2 construct measured by MTT assay (%, Mean±SD) Day

C6

LXSN

AS1

AS2

AS3

F

p

1 2 3 4 5 6

100 100 100 100 100 100

99.77±0.89 99.42±0.86 99.38±0.67 99.38±0.67 99.78±0.63 99.98±0.81

98.97±0.56 95.80±0.47 77.04±1.15 65.27±0.73 57.56±0.54 46.91±0.56

93.37±0.91 87.84±0.89 72.37±0.52 60.34±0.64 47.10±0.61 37.61±0.85

94.99±0.95 88.96±1.68 72.71±0.64 59.74±1.69 54.48±1.33 41.27±1.06

8.558 25.361 50.081 207.75 183.032 1157.30

>0.05 0.001 0.001 0.001 0.001 0.001

Note: C6: parental C6 cells; LXSN: C6 cells transfected with empty vector; AS1–3: Three C6 cell clones transfected with antisense AKT2 construct. Day 1–6: C6 compared with LXSN, p>0.05; AS1–3 compared with C6 and LXSN, p<0.001. Table 2. Proliferative activity analysis of C6 cell line transfected with antisense-AKT2 construct using PCNA staining

Table 3. Cell cycle analysis of C6 cell line transfected with antisense AKT2 constructs by FCM

Cell line

PCNA LI (x±s)

Cell lines

G0+G1

S

G2+M

C6 LXSN AS1 AS2 AS3

85.83±4.49 85.00±5.51 65.50±3.83 66.17±3.97 65.67±3.98

C6 C6-LXSN AS

57.49 58.55 69.46

24.99 24.40 10.54

17.52 17.05 20.00

Note: C6-AS1–3 compared with C6 and LXSN, p<0.001(LSD).

upregulated in C6 cells transfected with AS–AKT2 RNA, implicating that the differentiation of malignant glioma cells was induced (Figure 4). Detection of apoptosis Using TUNEL method, there were nearly no apoptotic cells found in parental C6 cells or the cells transfected with empty vector. However, apoptosis was prominently increased in C6 cells transfected with AS–AKT2 RNA (Figure 5). General behavior and survival of animals The general condition of control C6 rats was deteriorated one week after receiving injection of parental C6 glioma cells. A reduction in drinking and eating, weight loss, and progressive left hemiplegia appeared. Except one rat was remained for trial of high doses of AS–AKT2 RNA treatment at the end of 2 weeks following implantation of C6 cells, all the remaining nine rats died within 20 days after implantation. The average survival time was 17.8±0.92 days. The general behavior of rats in treated control group was the same as

Figure 4. Western blot analysis of GFAP expression in C6 cells transfected with AS–AKT2 constructs. The GFAP expression is dramatically increased in AS1–3 cell clones.

those in control C6 group. The average survival time of ten rats was 17.5±1.1 days. There was no statistical difference at the survival time between control C6 and treated control groups. In transfected group, six rats had the similar general manifestations to those in control C6 group at the beginning of second week after injection. They died on day 22, 22, 25, 27, 28 and 28, respectively. However, the remaining four rats kept normal behavior continuously up to 60 days of observation period (Figure 6). Among the ten rats that were injected with C6 glioma cells at first and then treated with AS–AKT2 RNA intratumorally, five rats did not respond well and their general condition was deteriorated and died on day 18, 20, 25, 27 and 28, respectively. The remaining five rats had got a favorable response as shown by MRI and their general condition returned to normal status grad-

Figure 3. Flow cytometry analysis of C6 cells transfected with AS–AKT2 constructs. SPF (S phase fraction) is decreased in cells transfected with AS–AKT2 construct.

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Figure 5. In situ cell death detection of C6 cells transfected with AS–AKT2 constructs. Nearly no apoptotic cells were found in the C6 cells and cells transfected with empty vector. Apoptotic cells are prominently increased in AS–AKT2 transfected cells.

ually. Among them, one rat with a tiny residual tumor died of anesthesia after MRI examination on day 28 and another rat with small residual tumor was retreated with activated AKT2 (Myristoylated AKT2) RNA as described later. The remaining three rats kept alive up to 120 days (Figure 7).

In the rats of control C6 group and treated control group examined by MRI, the tumor foci begun to develop on day 5 after implantation and appeared as an enhanced distinct tumor foci on day 7. They grew rapidly and enlarged to occupy the most portion of the right cerebral hemisphere at the end of second week after implantation. They were homogenously enhanced or irregularly rim enhanced. Mean tumor volume measured on MRI at the end of second week in control C6 and treated control group was 182.64±58.8 mm3 and 198.67±22.75 mm3, respectively. The corresponding histopathological examination in both group of animals showed the active proliferation of C6 glioma cells with necrosis, hemorrhage and neovascularization in the tumor foci. There were single glioma cell infiltration and tiny tumor foci in the nearby brain parenchyma, and

small amount of glioma cells scattered in choroids plexus of lateral ventricle, subependymal region and subarachnoid space. Among the rats of transfected group examined by MRI, tumor did not develop in four rats that confirmed by pathological examination later. Tumor growth in the remaining rats was slower than those in the control group. The average tumor volume at the end of second week was 110.38±22.86 mm3. Six rats in this group died within 3–4 weeks after implantation and histopathological findings of their tumors did not show any significant differences with control rats. While the other four rats remaining alive were killed on day 60 after implantation and no tumor foci were found in brain parenchyma, only a small amount of glioma cells was distributed in subarachnoid space and choroid plexus of lateral ventricle. MRI features of the rats in treated group were the same as those in control rats during the first two weeks after implantation. Tumor foci were regressed and disappeared on MRI examination at fourth and eighth week after implantation in four rats (Figure 8). Histopathological findings of the rats died within 4 weeks after implantations were similar to the control rats, but the tumor volume was smaller. The three rats kept alive

Figure 6. Survival analysis of the rats in C6, LXSN and transfected groups by Log-rank method (Log-rank 18.82, p=0.0001).

Figure 7. Survival analysis of the rats in C6, LXSN and treated groups by Log-rank method (Log-rank 18.82, p<0.0001).

MRI and corresponding histopathological findings

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Figure 8. Dynamic MRI scan of a rat in the treated group. MRI scan at 1, 2, 4, and 8 weeks after implantation of C6 glioma cells. Glioma gradually regresses and disappears eventually at the week 8 after implantation.

were sacrificed on day 120 after implantation and no tumor foci, but a few glioma cells in the subarachnoid space and choroids plexus of lateral ventricle were found. It should be mentioned that one rat with small residual tumor on MRI examination at fourth week after implantation was reinjected the plasmid LXSN– Myr-AKT2 DNA (4 lg)-lipofectamine complexes. The tumor showed regrowth to the original size on regular examination of MRI and the rat died in seventh week after treatment with Myr-AKT2 (Figure 9). The another interesting case also should be mentioned is that one control rat with critical general status, left hemiplegia and large tumor volume (185.23 mm3) was tried to treat with high doses of plasmid LXSN–AS– AKT2 DNA–lipofectamine complexes by both intratumoral and intramuscular injection (20 lg plasmid DNA for each site). By MRI monitoring, it was found that the tumor was significantly regressed two week after treatment and disappeared at the end of eighth week after treatment (Figure 10). The general behavior and hemiplegia returned to normal as well. Immunostaining for AKT2, PCNA, cyclin Dl, MMP2/9 expression in homograft tumor tissue The expression of AKT2, MMP2/9, cyclinD1, GFAP, PCNA was examined in tumor specimens of the rats naturally died in different groups, which included nine rats from control C6 group, ten rats from treated con-

trol group, five rats from transfected group, five rats from treated group and one additional rat from treated group reinjected with myristoylated AKT2. AKT2 expression was homogenous in tumor tissues of control C6 and treated control rats, while the surrounding normal brain tissues had no expression. The intensity and the positive staining cells of AKT2 expression were significantly reduced in tumor specimens from the rats of transfected and treated groups. However, its expression was inhibited more intensively in the center of the tumor nearby to the intratumoral injection site of AS–AKT2 complex than the peripheral portion of the tumor. Either the intensity or the number of positive staining cells of AKT2 expression in tumor specimen from one rat retreated with Myr-AKT2 RNA was similar to that of the control rats. The expression of PCNA, MMP2/9, cyclin D1 was also greatly reduced in tumor tissues of the rats in transfected and treated groups as compared with that in control rats (Figure 11, I–IV). There were nearly no apoptotic cells found in the tumor tissues of the control rats while a large amount of apoptotic cells could be found in the gliomas of the transfected and treated groups (Figure 11, V). The apoptotic cells were evenly distributed in gliomas of transfected rats, but mostly aggregated in the area nearby the intratumoral injection site with AS–AKT2 in tumors of treated group. In tumor of Myr-AKT2 retransfected rat, only a few apoptotic cells were scattered in the periphery of tumor.

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Figure 9. Activated AKT2 (Myr-AKT2) construct is reinjected intratumorally into a rat with a small residual tumor in the treated group, as demonstrated by MRI scan at fourth week after implantation. The tumor regrows to the fatal volume 7 weeks after retreatment as shown by MRI and histopathological examination of brain using H&E staining.

Discussion In the present study, we have used the antisense approach to investigate the oncogenic role of AKT2 in the development of malignant gliomas and the effects of

antisense AKT2 RNA on the inhibition of glioma cell growth. We have demonstrated that antisense AKT2 RNA is effective in inhibiting the elevated AKT2 expression in C6 glioma cells both in vitro and in vivo, Following the transfection with antisense AKT2 RNA,

Figure 10. One control rat with large volume of tumor 2 weeks after transplantation of C6 glioma cells, as shown by MRI, is treated with high doses of AS–AKT2 construct. The tumor disappears eventually at week 8 of treatment.

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Figure 11. Immunohistochemical staining of AKT2, MMP9, GFAP, cyclin D1 expression and apoptosis in tumor tissues of the rats in (a) control, (b) transfected, and (c) treated groups. (400).

the growth rate and proliferation activity of C6 glioma cells are significantly inhibited. GFAP expression of C6 cells is upregulated and cell apoptosis is induced in vitro.

These observations indicate that antisense AKT2 RNA is able to reverse the malignant phenotype of C6 glioma cells. The in vivo study shows that the tumorigenicity of

9 C6 glioma cells is evident. Ten rats for each of the control C6 and treated control groups developed a large tumor and died within 3 weeks after implantation. However, the growth of tumors in the six rats of transfected group was slow than the tumors of the control rats, they died with a prolonged period and the remaining rats failed to form tumors due to lowering of tumorigenicity of transfected cells. Among the ten rats bearing C6 gliomas treated with antisense AKT2 RNA, tumor growth was not inhibited in five rats, but the rats died with a longer period of survival than the control rats, while the other five rats were treated successfully to suppress the tumor growth. Except one rat retreated with Myr-AKT2 RNA, the tumors of the remaining rats disappeared eventually. Three of them were kept alive up to 120 days without recurrence of tumors on histopathological examination. We have demonstrated further the role of AKT2 in two special cases mentioned above. The first is that one control rat bearing large tumor in a critical condition is treated with high doses of antisense AKT2 RNA and dramatically recovered to normal. On the contrary, the other one rat bearing a small residual tumor after treatment with antisense AKT2 RNA is reinjected MyrAKT2 RNA intratumorally and the regressed tumor is regrown to the fatal volume. These findings not only reveal the important role of AKT2 in gliomagenesis, but also indicate that treatment with an optimal dose of antisense RNA is crucial for reaching favorable response. In addition to the inhibition of tumor growth by antisense AKT2 RNA, the expression of cell cycle driver, cyclin D, and the most importantly, the extracellular matrix degrading enzymes MMP2/9, which have been found to highly correlate with the invasion potential of glioma cells [23,39–41], is significantly inhibited, while the expression of GFAP, the marker associated with the growth and differentiation of astrocytic gliomas [42,43], is upregulated in AS–AKT2 transfected or treated tumor cells. These evidences further identify both positively and negatively that AKT2 contributes to the development and progression of tumors. The report on antisense AKT2 approach directed against malignant glioma cells has not been found yet. However, Narita et al. [44] demonstrated that transfection of a kinase-dead dominant-negative AKT mutant into UM87 glioma cells with mutant EGFR expression results in downregulation of tumorigenicity and upregulation of cyclin-dependent kinase p27. Treatment of cancer cells with overexpression of AKT2 by antisense AKT2 RNA has only been reported by Cheng et al. [30] which shows that the growth, invasiveness and tumorigenicity of pancreatic cancer cells with overexpression of AKT2 are greatly reduced after treatment with antisense AKT2 RNA and the pancreatic cells without elevated expression of AKT2 appear to be refractory to antisense AKT2 RNA treatment. This result is similar to our findings in malignant glioma cells in vivo. Since most of the highly malignant gliomas overexpressing growth factors and their receptors, especially EGFR and about 30% glioblastomas have mutated PTEN which negatively regulates the AKT activity

under normal situation [45–47]. Therefore, the PI3K/ AKT signal transduction cascade is activated in many, if not all glioblastomas, and it may be a suitable target for therapeutic intervention in gliomas. However, glioma, just like the tumors in other sites of the body, is a disease involved with multiple genes, anti-AKT strategy may be more effective in combination with other molecular therapies targeting genes important to the pathogenesis of gliomas, such as EGFR, PTEN, p53, etc and some synthetic inhibitors, including EGFR inhibitors (such as ZD1839 or Iressa) [48,49] and PI3K inhibitors (such as CCI-779, RAD-001) [49]. Therefore, further studies are necessary to develop better therapeutic strategies of malignant gliomas. Even though the antitumor effect we observed using AKT2 antisense approach may be due to the downregulation of AKT2-mediated downstream target signaling proteins. However, a potential alternative explanation of loss of tumorigenicity has been raised by the observation of Trojian’s work using similar model and approach targeting the IGF-I, a growth factor which is highly expressed in glioma cells [50]. They observed that subcutaneous injection of parental C6 glioma cells or cells transfected with empty vectors resulted in the development of large tumors after 2 weeks. However, rats injected with C6 cells transfected with antisense IGF-I showed no tumors after 40 weeks of observation. They also observed that a small cyst was apparent in six rats after two weeks of injection of antisense IGF-I transfectant cells. Histologic examination revealed a few glioma cells infiltrated by a large number of mononuclear cells in rats injected with antisense IGF-I transfectant cells. Similar observation was not found in rats injected with parental C6 cells, suggesting that parental cells may escape the host immune response. In a separate study, they further showed that the antitumor effects result from a glioma-specific immune response involving CD8+ lymphocytes and antisense blocking of IGF-I expression may reverse a phenotype that allows C6 glioma cells to evade the immune system [51]. Therefore, whether the loss of tumorigenicity is due in part to the enhanced host immune response to AKT2 antisense transfectant cells, remains to be studied further to examine this possibility.

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