Additive effects of the combined expression of soluble forms of GAS1 and PTEN inhibiting glioblastoma growth.

The overexpression of GAS1 (Growth Arrest Specific 1) in glioma cells induces cell cycle arrest and apoptosis. We previously demonstrated that the apoptotic process set off by GAS1 is caused by its capacity to inhibit the Glial cell-derived neurotrophic factor (GDNF)-mediated intracellular survival signaling pathway. Whereas on the other hand, PTEN is a tumor suppressor, inactive in many tumors, and both GAS1 and PTEN inhibit the PI3K/AKT pathway. Therefore, it is relevant to investigate the potential additive effect of the overexpression of GAS1 and PTEN on tumor growth. In particular, we employed secreted forms of both GAS1 (tGAS1) and PTEN (PTEN-LONG, or PTEN-L) and tested their combined effect on glioma cells. We observed that the co-expression of both the proteins inhibited the growth of U-87 MG human glioblastoma cells more effectively than when independently expressed, and decreased the activity of both AKT and ERK1/2. Interestingly, the combination of the soluble forms was always the most effective treatment. To improve the transfer of tGAS1 and PTEN-L, we employed a lentiviral vector with a p2A peptide-enabled dual expression system that allowed the generation of the two proteins using a single promoter (CMV), in equimolar amounts. The viral vector reduced the growth of U-87 MG cells in vitro and had a striking effect in inhibiting their proliferation after inoculating it into the immunosuppressed mice. The present results support a potential adjuvant role for the combined use of tGAS1 and PTEN-L in the treatment of glioblastoma.

Exogenous induction of unphosphorylated PTEN reduces TGFß-induced extracellular matrix expressions in lung fibroblasts.

Transforming growth factor ß (TGFß) plays an important role in regulating aberrant extracellular matrix (ECM) production from alveolar/epithelial cells (AECs) and fibroblasts in pulmonary fibrosis. Although the tumor suppressor gene phosphatase and tensin homologue deleted from chromosome 10 (PTEN) can negatively control many TGFß-activated signaling pathways via the phosphatase activity, hyperactivation of the TGFß-related signaling pathways is often observed in fibrosis. Loss of PTEN expression might cause TGFß-induced ECM production. In addition, TGFß was recently shown to induce loss of PTEN enzymatic activity by phosphorylating the PTEN C-terminus. Therefore, we hypothesized that exogenous transfer of unphosphorylated PTEN (PTEN4A) might lead to reduce TGFß-induced ECM expression in not only epithelial cells but also fibroblasts. Adenovirus-based exogenous PTEN4A induction successfully reduced TGFß-induced fibronectin expression and retained ß-catenin at the cell membrane in human epithelial cells. Exogenous unphosphorylated PTEN also attenuated TGFß-induced ECM production and inhibited TGFß-induced ß-catenin translocation in a human fibroblast cell line and in mouse primary isolated lung fibroblasts. Conversely, TGFß-induced α-smooth muscle actin expression did not seem to be inhibited in these fibroblasts. Our data suggest that exogenous administration of unphosphorylated PTEN might be a promising strategy to restore TGFß-induced loss of PTEN activity and reduce aberrant TGFß-induced ECM production from epithelial cells and fibroblasts in lung fibrosis as compared with wild-type PTEN induction.

Precise therapeutic effect of self-assembling gold nanocluster-PTEN complexes on an orthotropic model of liver cancer.

PURPOSE: Presently, liver cancer is still one of the malignant tumors with high mortality. As far as the treatment of liver cancer is concerned, the most effective method is still liver transplantation. But every year, many liver cancer patients die from the lack of a proper liver transplant, or from waiting for a liver transplant. Therefore, it is very important to find new and effective treatment for patients with liver cancer. METHODS: Herein, the cell model and the orthotropic liver tumor mice model have been performed to verify the results of our treatment. We found that the in situ synthesized gold nanocluster-PTEN (GNC-PTEN) complexes can effectively target and realize the fluorescence imaging of the liver tumor. RESULTS: GNC-PTEN complexes could inhibit the proliferation, invasion, and metastasis of liver cancer cells. And the results also showed that GNC-PTEN complexes could be well targeted liver tumor at 6 h and the liver tumor in mice group treated with GNC-PTEN complexes almost disappeared. CONCLUSION: This is a simply and effectively method to realize liver cancer imaging and inhibition. This may raise the possibility for the accurate image/diagnosis and simultaneously efficient treatment of liver cancer in the relevant clinic application.

Self-responsive co-delivery system for remodeling tumor intracellular microenvironment to promote PTEN-mediated anti-tumor therapy.

Delivering the pten gene into tumor cells to reacquire PTEN functionality is considered to be an attractive method for cancer treatment. However, the inhibition effect of the tumor intracellular microenvironment (TIME), especially at the high reactive oxygen species (ROS) level, on pten expression and PTEN protein functionality was nearly overlooked. Herein, the development of a potential strategy is described, which enhances PTEN-mediated anti-tumor capability by exhausting the intracellular ROS in TIME. To achieve this, poly(ethyleneimine) (PEI)-modified DSPE was introduced to protect the pten plasmid, and form liposomes for encapsulating the "scavenger" of oxidation homeostasis, epigallocatechin-3-gallate (EGCG). Notably, this was a simple system with improved safety compared which when compared with the use of PEI could accomplish efficient pten transfection and simultaneous disintegration to cause transient release of EGCG responding to the endosome environment through the "proton sponge effect". In the cytoplasm, EGCG depleted ROS and promoted the expression of the pten gene as well as restoring protein functionality, thus negatively regulating the PI3K-AKT signaling pathway. In vitro and in vivo results revealed that this system significantly inhibited tumor growth via remodeling of the TIME, and provided a promising way to control malignant tumors.

A Novel Rat Model of Nonalcoholic Fatty Liver Disease Constructed Through CRISPR/Cas-Based Hydrodynamic Injection.

CRISPR/Cas technology has been widely used in generating conventional and conditional gene knockout animals through germline mutation. A recent study has demonstrated that CRISPR/Cas system also worked on nongermline mutation in mice liver via hydrodynamic injection of vector plasmid to blood circulation. However, whether this is also applied to rat and what is the optimal concentration of vector plasmid still need to be studied. Here, we attempted to use pX330-Pten plasmid to knock down the expression of Pten in rat liver for generation of nonalcoholic fatty liver disease (NAFLD) rat model. A range of pX330-Pten dosages (75, 150 and 300 µg/100 g) in 10 ml of saline water was injected into the blood circulation of SD rats within 30 s by tail vein injection. The data showed that, 9 weeks after injection, no differences of TC, AST and ALT were found between control and the treatment groups (low-dose, mid-dose, high-dose and fat-diet groups). However, the high-dose group revealed severe lipid deposition and significant knockdown of Pten, which was validated by western blot and real-time PCR. Notably, lipid deposition and Pten knockdown were detected in the liver of animals from the high-dose group as early as 6 weeks after injection. Taken together, our findings demonstrated that hydrodynamic injection of pX330-Pten plasmid at the dosage of 300 µg/100 g can knock down the expression of Pten in rat liver efficiently, resulting in lipid deposition as well as NAFLD. Taken together, this study presents a novel and efficient method to edit genome in rat liver.

PTEN and TRAIL genes loaded zein nanoparticles as potential therapy for hepatocellular carcinoma.

Gene therapy is one of the recent approaches in treatment of hepatocellular carcinoma (HCC). Development of a vector or vehicle that can selectively and efficiently deliver the gene to target cells with minimal toxicity is an urgent demand. In the present study, phosphatase and tensin homolog (PTEN) and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) genes were loaded to zein nanoparticles (ZNPs). The formulated PTEN and TRAIL-loaded ZNPs were tested for their in vitro and in vivo potential antitumor efficacy using liver tumor cells (HepG2) and HCC-induced rats as animal model. Also, mRNA expression of p53, VGEF and MMP-2 were carried out as markers of apoptosis, angiogenesis and metastasis in animal liver tissues. The results of the study showed that both PTEN and TRAIL-loaded ZNPs proved anti-proliferative activity against HepG2 cell lines with IC50 values of 0.09, 0.25 µg/ml, respectively. In vivo assay confirmed decrease in mRNA expression of both VEGF and MMP-2 with increased in P53 expression level in liver tissues of the treated animals. Therefore, authors introduced new integration between gene therapy and nanotechnology in the form of PTEN and TRAIL-loaded ZNPs that proved potential to be used in gene therapy for the treatment of HCC.

Intracellular delivery of the PTEN protein using cationic lipidoids for cancer therapy.

The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a tumor suppressor, mutated or inactive in a large percentage of human cancers. Restoring PTEN activity in cancer cells through gene therapy has shown to inhibit cell growth and induce apoptosis, particularly in cells with a PTEN deficiency. Gene therapy, however, comes with some inherent risks such as triggering an immune response and permanent off target effects. Nanoparticle assisted protein delivery could mitigate these liabilities while maintaining therapeutic integrity. In this report, we evaluated the use of cationic lipid-like (lipidoid) materials to intracellularly deliver the PTEN protein. We synthesized a small library of cationic lipidoid materials and screened for the delivery of PTEN based on cell viability. The lipidoid material EC16-80 was selected for high efficacy and the subsequent lipidoid-protein complex was characterized using DLS, zeta potential, and TEM. Intracellular delivery of PTEN with EC16-80 to the PTEN deficient prostate cancer cell line PC-3 resulted in a significant decrease in activated AKT and induced apoptosis. Interestingly, delivery of PTEN to PTEN deficient prostate cancer cell lines PC-3 and LNCaP compared to the breast cancer cell line, MCF-7 with endogenous PTEN, resulted in significantly lower IC50 values in PC-3 and LNCaP cells indicating that the treatment is predominantly specific to PTEN-deficient cells. Altogether, these results demonstrate the first intracellular delivery of recombinant PTEN using a synthetic delivery vehicle and highlight the potential of intracellular PTEN protein delivery as a potential targeted cancer therapy.

Preventive effects of AdR-siPTEN through the regulation of NMDA receptor NR2B subunit in trigeminal ganglia of migraine rats.

OBJECTIVE: Migraine is a refractory disease that is due to neuronal hyperexcitability, and has high incidence, mortality, and disability rates. The N-methyl-D-aspartate receptor 2B (NR2B) subunit has been found to play an important role in the pathogenesis of migraine. There is evidence suggesting that a tumor suppressor phosphatase and tensin homolog (PTEN) can confer a neuroprotective effect on cerebral ischemic injury by regulating NR2B levels. However, the role of PTEN in migraines is still unclear. This study aimed to define whether PTEN is involved in the pathogenesis of migraine through modulating NR2B, nitric oxide synthase (NOS), and nitric oxide (NO) in the trigeminal ganglia of rats with glyceryl trinitrate-induced migraine. METHODS: Adenovirus-expressing siPTEN or RFP was independently injected into the Sp5 (spinal trigeminal nucleus) of rats suffering from migraines. Seven days later, tactile sensory testing was performed to detect the tactile threshold. Immunofluorescence assay, western blot assay, RT-PCR, and biochemical examination were done to measure PTEN, NR2B, NOS, and NO levels in the trigeminal ganglia of migraine rats. RESULTS: NR2B, NOS, and NO levels significantly (P<0.05) decreased in the trigeminal ganglia of migraine-affected rats pretreated with adenovirus-expressing siPTEN. CONCLUSION: These results suggest that PTEN in trigeminal ganglia is implicated in the pathogenesis of migraine, and PTEN may be a novel and promising candidate for future treatment and/or prevention of migraine via regulating NR2B and decreasing NO production in trigeminal ganglia.

Effect of wild type PTEN gene on proliferation and invasion of multiple myeloma.

We explored the effect of the wild type PTEN gene on the proliferation, apoptosis and invasive ability of multiple myeloma (MM) cells from MM patients and RPMI 8226 cells (a human myeloma cell line), and the effect of the PTEN/focal adhesion kinase (FAK)/MMP signaling pathway on the invasion activity of RPMI 8226 cells. The proliferation of RPMI 8226 cells and purified myeloma cells from MM patients were markedly inhibited after these cells were transfected with recombinant adenovirus-PTEN vectors containing green fluorescent protein (Ad-PTEN-GFP). Maximum growth inhibition of RPMI 8226 cells and purified myeloma cells from MM patients by AD-PTEN-GFP was 42.01 and 24.75%, respectively. After transfection with PTEN-siRNA, the proliferation of RPMI 8226 cells was increased significantly compared with NS-siRNA transfected controls. The maximal survival rate was 141.55 +/- 8.34% in PTEN-siRNA transfected RPMI 8226 cells. Apoptosis of RPMI 8226 cells or purified myeloma cells from MM patients in the Ad-PTEN-GFP group was increased significantly when compared with that in the Ad-GFP (adenovirus vectors only expressing green fluorescent protein) group (p < 0.01). The cell cycle of RPMI 8226 cells was arrested at the G2/M phase. Furthermore, the number of cells that migrated through the matrigel and filter from the upper chamber to the lower chamber in the transwell assay in the Ad-GFP group was significantly larger than that in the Ad-PTEN-GFP group (52.65 +/- 7.39 vs. 23.50 +/- 6.12, p < 0.01). In the PTEN-siRNA group, the cell number (79.50 +/- 11.89) was significantly larger than that in the NS-siRNA group (47.17 +/- 7.76, p < 0.01). When RPMI 8226 cells were transfected with Ad-PTEN-GFP or NS-siRNA, the expression level of PTEN mRNA was up-regulated, and the expression levels of FAK, MMP-2 and MMP-9 mRNA were down-regulated significantly compared with that of the Ad-GFP group and the PTEN-siRNA group (p < 0.01, p < 0.05). The protein levels of FAK and p-FAK, MMP-2 and MMP-9 in RPMI 8226 cells which were transfected with Ad-PTEN-GFP decreased significantly, but increased significantly in PTEN-siRNA transfected RPMI 8226 cells (p < 0.01, p<0.05). These results indicated that wild type PTEN, which inhibited FAK, MMP-2, and MMP-9, could suppress the proliferation and invasion ability of multiple myeloma cells. Modulating the expression of PTEN may be a potential strategy for the treatment of multiple myeloma.

Urocanic acid-modified chitosan-mediated PTEN delivery via aerosol suppressed lung tumorigenesis in K-ras(LA1) mice.

The low efficiency of conventional therapies in achieving long-term survival of lung cancer patients calls for development of novel options. Revisiting of aerosol gene delivery may provide an alternative for safe and effective treatment for lung cancer. In this study, imidazole ring-containing urocanic acid-modified chitosan (UAC) designed in the previous study was used as a gene carrier. The potential effects of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) on Akt-related signals and cell cycle regulation were evaluated. Aerosols of UAC-PTEN were delivered into K-ras(LA1) lung cancer model mice through the nose-only inhalation system twice a week for total 4 weeks. Delivered PTEN suppressed lung tumor development significantly through nuclear complex formation between PTEN and p53, suppressing Akt-related signals as well as cell cycle regulation. Together, our results suggest that aerosol delivery of UAC-PTEN may be compatible with noninvasive in vivo gene therapy.