Lentiviral vector-mediated shRNA against AIMP2-DX2 suppresses lung cancer cell growth through blocking glucose uptake.

Aminoacyl-tRNA synthetases [ARS]-interacting multifunctional protein 2 (AIMP2) has been implicated in the control of cell fate and lung cell differentiation. A variant of AIMP2 lacking exon 2 (AIMP2-DX2) is expressed in different cancer cells. We previously studied the expression level of AIMP2-DX2 in several lung cell lines and reported elevated expression levels of AIMP2-DX2 in NCI-H460 and NCI-H520. Here, we report that the suppression of AIMP2-DX2 by lentivirus mediated short hairpin (sh)RNA (sh-DX2) decreased the rate of glucose uptake and glucose transporters (Gluts) in NCI-H460 cells. Down-regulation of AIMP2-DX2 reduced glycosyltransferase (GnT)-V in the Golgi apparatus, while inducing the GnT-V antagonist GnT-III. Down-regulation of AIMP2-DX2 also suppressed the epidermal growth factor receptor/mitogen activated protein kinase (EGFR/MAPK) signaling pathway, leading to the decrease of the proliferation marker Ki-67 expression in nuclei. Furthermore, dual luciferase activity reduced capdependent protein translation in cells infected with sh-DX2. These results suggest that AIMP2-DX2 may be a relevant therapeutic target for lung cancer, and that the sh-DX2 lentiviral system can be an appropriate method for lung cancer therapy.

Suppression of tumor growth in xenograft model mice by small interfering RNA targeting osteopontin delivery using biocompatible poly(amino ester).

Gene therapy using small interfering RNA (siRNA) is a novel strategy for effective anticancer therapies. However, low gene transfection efficiency and technical difficulties linked to siRNA delivery limit their practical application for gene delivery. Therefore, development of effective siRNA carriers is required. Overexpression of osteopontin (OPN) and its association with tumorigenesis has been reported in a majority of breast cancers. In this study, we used siRNA against OPN (siOPN) and investigated the possible OPN-dependent signaling pathway and the potential use of poly(amino ester) (PAE) based on glycerol propoxylate triacrylate (GPT) and spermine (SPE) for siRNA delivery. The GPT-SPE could effectively condense siRNA and protect the siRNA from RNaseA enzyme degradation. GPT-SPE/siRNA complexes showed good intracellular uptake and high gene silencing efficiency in vitro. Furthermore, in the breast cancer xenograft model, intratumoral injection of GPT-SPE/siOPN significantly inhibited tumor growth. These results demonstrated that silencing of OPN effectively suppressed the growth of breast cancer cells and further suggested that delivery of siRNA using GPT-SPE may act as an effective gene carrier for cancer therapy.

Low dietary inorganic phosphate affects the lung growth of developing mice.

Inorganic phosphate (Pi) plays a critical role in diverse cellular functions, and regulating the Pi balance is accomplished by sodium-dependent Pi co-transporter (NPT). Pulmonary NPT has recently been identified in mammalian lungs. However, to date, many of the studies that have involved Pi have mainly focused on its effect on bone and kidney. Therefore, current study was performed to discover the potential effects of low Pi on the lung of developing transgenic mice expressing the renilla/firefly luciferase dual reporter gene. Two-weeks old male mice divided into 2 groups and these groups were fed either a low PI diet or a normal control diet (normal: 0.5% Pi, low: 0.1% Pi) for 4 weeks. After 4 weeks of the diet, all the mice were sacrificed. Their lungs were harvested and analyzed by performing luciferase assay, Western blotting, kinase assay and immunohistochemistry. Our results demonstrate that low Pi affects the lungs of developing mice by disturbing protein translation, the cell cycle and the expression of fibroblast growth factor-2. These results suggest that optimally regulating Pi consumption may be important to maintain health.

Synergistic effect of ERK inhibition on tetrandrine-induced apoptosis in A549 human lung carcinoma cells.

Tetrandrine (TET), a bis-benzylisoquinoline alkaloid from the root of Stephania tetrandra, is known to have anti-tumor activity in various malignant neoplasms. However, the precise mechanism by which TET inhibits tumor cell growth remains to be elucidated. The present studies were performed to characterize the potential effects of TET on phosphoinositide 3-kinase/Akt and extracellular signal-regulated kinase (ERK) pathways since these signaling pathways are known to be responsible for cell growth and survival. TET suppressed cell proliferation and induced apoptosis in A549 human lung carcinoma cells. TET treatment resulted in a down-regulation of Akt and ERK phosphorylation in both time-/concentration-dependent manners. The inhibition of ERK using PD98059 synergistically enhanced the TET-induced apoptosis of A549 cells whereas the inhibition of Akt using LY294002 had a less significant effect. Taken together, our results suggest that TET: i) selectively inhibits the proliferation of lung cancer cells by blocking Akt activation and ii) increases apoptosis by inhibiting ERK. The treatment of lung cancers with TET may enhance the efficacy of chemotherapy and radiotherapy and increase the apoptotic potential of lung cancer cells.

High dietary inorganic phosphate increases lung tumorigenesis and alters Akt signaling.

RATIONALE: Phosphate (Pi) is an essential nutrient to living organisms. Recent surveys indicate that the intake of Pi has increased steadily. Our previous studies have indicated that elevated Pi activates the Akt signaling pathway. An increased knowledge of the response of lung cancer tissue to high dietary Pi may provide an important link between diet and lung tumorigenesis. OBJECTIVES: The current study was performed to elucidate the potential effects of high dietary Pi on lung cancer development. METHODS: Experiments were performed on 5-week-old male K-ras(LA1) lung cancer model mice and 6-week-old male urethane-induced lung cancer model mice. Mice were fed a diet containing 0.5% Pi (normal Pi) and 1.0% Pi (high Pi) for 4 weeks. At the end of the experiment, all mice were killed. Lung cancer development was evaluated by diverse methods. MEASUREMENT AND MAIN RESULTS: A diet high in Pi increased lung tumor progression and growth compared with normal diet. High dietary Pi increased the sodium-dependent inorganic phosphate transporter-2b protein levels in the lungs. High dietary consumption of Pi stimulated pulmonary Akt activity while suppressing the protein levels of tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 as well as Akt binding partner carboxyl-terminal modulator protein, resulting in facilitated cap-dependent protein translation. In addition, high dietary Pi significantly stimulated cell proliferation in the lungs of K-ras(LA1) mice. CONCLUSIONS: Our results showed that high dietary Pi promoted tumorigenesis and altered Akt signaling, thus suggesting that careful regulation of dietary Pi may be critical for lung cancer prevention as well as treatment.

Chondroitin sulfate extracted from ascidian tunic inhibits phorbol ester-induced expression of Inflammatory factors VCAM-1 and COX-2 by blocking NF-kappaB activation in mouse skin.

Inflammatory factors are known to play a key role in promoting tumorigenesis; therefore, it is a promising strategy to inhibit the inflammation for cancer prevention. The current study was performed to investigate the potential effects of chondroitin sulfate (CS) extracted from ascidian tunic on the expression of inflammatory factors induced by treatment with 12- O-tetradecanoylphorbol-13-acetate (TPA) and to elucidate the underlying molecular mechanism of CS action in mouse skin inflammation. TPA was topically applied to the shaven backs of ICR mice with or without CS (1 or 2 mg) for 4 h. The results demonstrated that CS suppressed TPA-induced edema and reduced the expression of cyclooxygenase-2, vascular cell adhesion molecule-1, and Akt signaling in mouse skin. These studies suggest that CS from ascidian tunic may be developed as an effective natural anti-inflammatory agent.

High dietary inorganic phosphate enhances cap-dependent protein translation, cell-cycle progression, and angiogenesis in the livers of young mice.

Inorganic phosphate (P(i)) plays a key role in diverse physiological functions. Recent studies have indicated that P(i) affects Akt signaling through the sodium-dependent phosphate cotransporter. Akt signaling, in turn, plays an important role in liver development; however, the effects of high dietary P(i) on the liver have not been investigated. Here, we examined the effects of high dietary phosphate on the liver in developing mice. We found that high dietary P(i) increased liver mass through enhancing Akt-related cap-dependent protein translation, cell cycle progression, and angiogenesis. Thus careful regulation of P(i) consumption may be important in maintaining normal development of the liver.

Poly(ester amine)-mediated, aerosol-delivered Akt1 small interfering RNA suppresses lung tumorigenesis.

RATIONALE: The low efficiency of conventional therapies in achieving long-term survival of patients with lung cancer calls for the development of novel therapeutic options. Recent advances in aerosol-mediated gene delivery have provided the possibility of an alternative for the safe and effective treatment of lung cancer. OBJECTIVES: To demonstrate the feasibility and emphasize the importance of noninvasive aerosol delivery of Akt1 small interfering RNA (siRNA) as an effective and selective option for lung cancer treatment. METHODS: Nanosized poly(ester amine) polymer was synthesized and used as a gene carrier. An aerosol of poly(ester amine)/Akt1 siRNA complex was delivered into K-ras(LA1) and urethane-induced lung cancer models through a nose-only inhalation system. The effects of Akt1 siRNA on lung cancer progression and Akt-related signals were evaluated. MEASUREMENTS AND MAIN RESULTS: The aerosol-delivered Akt1 siRNA suppressed lung tumor progression significantly through inhibiting Akt-related signals and cell cycle. CONCLUSIONS: The use of poly(ester amine) serves as an effective carrier, and aerosol delivery of Akt1 siRNA may be a promising approach for lung cancer treatment and prevention.

Chondroitin sulfate extracted from the Styela clava tunic suppresses TNF-alpha-induced expression of inflammatory factors, VCAM-1 and iNOS by blocking Akt/NF-kappaB signal in JB6 cells.

Inflammatory mediators are known to play a key role in tumorigenesis, therefore, it is a promising strategy to inhibit the inflammation for cancer prevention and/or treatment. Current study was performed to investigate the effects of chondroitin sulfate (CS) extracted from Styela clava tunic on TNF-alpha-induced inflammation and to elucidate the mechanism of CS on the regulation of inflammatory factors in JB6 cells. Our results showed that CS inhibited TNF-alpha-induced NF-kappaB activation and subsequent vascular cell adhesion molecule 1 and inducible nitric oxide synthase expressions by blocking Akt signals in JB6 cells. Our results suggest that CS may be developed as an effective anti-inflammatory agent in the future.

Serum immunoglobulin fused interferon-alpha inhibited tumor growth in athymic mice bearing colon 26 adenocarcinoma cells.

Interferon (IFN) has therapeutic potential for a wide range of infectious and proliferative disorders. However, the half-life of IFN is too short to have a stable therapeutic effect. To overcome this problem, serum immunoglobulin has been fused to IFN. In this study, the efficacy of serum immunoglobulin fused INFs (si-IFN1 and si-IFN2) was evaluated on athymic mice bearing colon 26 adenocarcinoma cells. Seven days after the implantation of tumor cells, each group of mice was injected once a week with si-IFN1 and si-IFN2 at two different concentrations (10 x : 30 microg/kg and 50 x : 150 microg/kg). A slight anti-tumoral effect was observed in all 10 x groups compared to the control. In the 50 x groups, however, si-IFN1 and si-IFN2 showed significant anti- tumoral effects compared to the control. To gain more information on the mechanisms associated with the decrease of tumor size, a Western blot assay of apoptosis-related molecules was performed. The protein expression of cytochrome c, caspase 9, 6, and 3 were increased by si-IFN1 and si-IFN2. These 2 IFNs also increased the expressions of p53, p21, Bax and Bad. Interestingly, si-IFN1 and si-IFN2 decreased the expression of VEGF-beta. Taken together, serum immunoglobulin fused IFNs increased therapeutic efficacy under current experimental condition.

High dietary inorganic phosphate affects lung through altering protein translation, cell cycle, and angiogenesis in developing mice.

Inorganic phosphate (Pi) plays a key role in diverse physiological functions. Several studies indicate that Pi may affect lung cell development through Na/Pi cotransporter (NPT). Several NPT subtypes have been identified in mammalian lung, and considerable progress has been made in our understanding of their function and regulation. Therefore, current study was performed to elucidate the potential effects of high dietary Pi on lungs of developing mice. Our results clearly demonstrate that high dietary Pi may affect the lung of developing mice through Akt-related cap-dependent protein translation, cell cycle regulation, and angiogenesis. Our results support the hypothesis that Pi works as a critical signal molecule for normal lung growth and suggest that careful restriction of Pi consumption may be important in maintaining a normal development.