MST1/2 inhibitor XMU-MP-1 alleviates the injury induced by ionizing radiation in haematopoietic and intestinal system.

The Hippo signalling pathway has been considered as potential therapeutic target in self-renewal and differentiation of stem and progenitor cells. Thus, mammalian Ste20-like kinase 1/2 (MST1/2) as the core serine-threonine kinases in the Hippo signalling pathway has been investigated for its role in immunological disease. However, little information of MST1/2 function in bone marrow suppression induced by ionizing radiation was fully investigated. Here, we reported that MST1/2 inhibitor XMU-MP-1 could rescue the impaired haematopoietic stem cells (HSCs) and progenitor cells (HPCs) function under oxidative stress condition. Also, XMU-MP-1 pretreatment markedly alleviated the small intestinal system injury caused by the total body irradiation 9 Gy and extended the average survival days of the mice exposed to the lethal dose radiation. Therefore, irradiation exposure causes the serious pathological changes of haematopoietic and intestinal system, and XMU-MP-1 could prevent the ROS production, the haematopoietic cells impairment and the intestinal injury. These detrimental effects may be associated with regulating NOX/ROS/P38MARK pathway by MST1/2.

Targeting of NHERF1 through RNA interference inhibits the proliferation and migration of metastatic prostate cancer cells.

The present study aimed to investigate the effects of Na+/H+ exchanger regulatory factor 1 (NHERF1) gene knockdown, using short-hairpin RNA (shRNA), on the malignant behaviors of prostate cancer cells. A pSuper.puro NHERF1 shRNA vector was transfected into PC-3M prostate cancer cells using Lipofectamine 2000. Stable cell lines were obtained and NHERF1 knockdown was verified through western blot analysis. MTT assays were then used to measure PC-3M cell proliferation; in addition, cell migration was assessed using a wound healing assay. Flow cytometry was employed in order to determine the effects of NHERF1 knockdown on apoptosis. Expression levels of apoptotic pathway proteins B cell lymphoma-2 (Bcl-2) and Bcl-2-associated X protein were then determined by western blot analysis. The results demonstrated that shRNA knockdown of NHERF1 significantly suppressed the proliferation of PC-3M cells by >50%. In addition, knockdown of NHERF1 significantly inhibited the migration of PC-3M cells. PC-3M cells harboring NHERF1 shRNA exhibited significantly increased apoptosis, with an ~4-fold increase compared with that of the parental PC-3M cells and cells transfected with an empty vector. Furthermore, the results revealed that knockdown of NHERF1 reduced the protein expression of Bcl-2, although the expression of Bax was unaltered. In conclusion, NHERF1 knockdown using shRNA inhibited the proliferation and migration of PC-3M cells and promoted apoptosis, highlighting the role of NHERF1 in prostate cancer progression.

Exploration of peptide T7 and its derivative as integrin αvß3-targeted imaging agents.

OBJECTIVE: The aim of the present study was to develop potential candidates of integrin αvß3-targeted imaging agent, which can facilitate the diagnosis and treatment of malignant solid tumors. METHODS: Peptides derived from tumstatin, named T7 and T7-6H, were derivatized to contain histidine in the C-terminus of their sequence and were labeled with (99m)Tc via nitrido and carbonyl precursors. The radiochemical purity and stability of (99m)Tc-labeled T7 and T7-6H were characterized by thin-layer chromatography. The whole body biodistribution was studied in NCI-H157-bearing BALB/c nude mice. RESULTS: The (99m)Tc-labeled T7 and T7-6H showed adequate in vitro stability, with a high radiochemical purity of over 90%. The dissociation constant (Kd) value of the (99m)Tc-labeled T7 and T7-6H ranged from 68.5 nM to 140.8 nM in U251 and NCI-H157 cell lines. (99m)Tc-labeled T7 and T7-6H showed no significant difference of biodistribution in mice. Furthermore, both T7 and T7-6H exhibited a poor blood-brain barrier penetration and a transient accumulation in lung; the uptake in tumor tissues was significantly higher than in muscle tissue, with a ratio of 5.8. CONCLUSION: (99m)Tc-labeled T7 and T7-6H can be regarded as promising single-photon emission computed tomography probes for imaging integrin αvß3, and need to be further studied for noninvasive detection of tumors.

[Enzyme-amplified time-resolved fluorescence detection for nucleic acid hybridization assays].

OBJECTIVE: To develop a new nonisotopic detection method of enzyme-amplified time-resolved fluorescence (EATRF) or enzyme-amplified lanthanide luminescence (EALL) for nucleic acid hybridization assays, which can be applied extensively in clinical diagnosis. METHODS: The method combines the high affinity of biotin-streptavidin system, amplification of enzyme, and inherent advantage of lanthanide chelate with the background elimination of time-resolved fluorescence detection. The conversion of 5-fluorosalicyl phosphate to 5-fluorosalicylic acid (5-FSA) by alkaline phosphatase. The salicylic acid product forms a luminescent ternary chelate with Tb3+ and EDTA. RESULTS: The dynamic range of the standard curve of EATRFA for nucleic acid hybridization assay was very wide, the range was more than third order of magnitude. The detection sensitivity was about 10 pg of target sequence. When the known target sequence was 20, 10 and 2 ng, the ratio of measured amount to known amount was 110%, 90% and 115% respectively. The main experimental conditions, for example, the irradiating time of ultraviolet rays, the concentrations of biotinylated probe, AP-SA, 5-FSAP and Tb-EDTA and the methods of washing in the related steps, have been optimized. A new stable technology of fluorescence has been developted. CONCLUSIONS: EATRF detection for nucleic acid hybridization assays is a new sensitive simple method, which has a great prospect.

[Application of target peptide in siRNA delivery 
for the research of lung cancer therapy].

Lung cancer is considered a kind of malignant tumors of the world highest incidence. As it is not sensitive to chemotherapy and easy to produce drug resistance, improving effect of anticancer drug becomes a research focus recent years. siRNA, small interfering RNA, can silence complemenary mRNA which is a kind of gene therapy. Target peptides are small molecular peptides which specifically bind to tumor surface materials. When used with siRNA, target peptides can increase accumulation of siRNA in tumor cells and enhance the silencing effect. As result, drug resistance of lung cancer reduced and the effect of therapy can be improved. This method provides new direction and strategy for targeted therapy of lung cancer. This article will make a brief overview of target peptides applying in siRNA dilivery for the research of lung cancer treatment.

Novel tumor-targeting, self-assembling peptide nanofiber as a carrier for effective curcumin delivery.

The poor aqueous solubility and low bioavailability of curcumin restrict its clinical application for cancer treatment. In this study, a novel tumor-targeting nanofiber carrier was developed to improve the solubility and tumor-targeting ability of curcumin using a self-assembled Nap-GFFYG-RGD peptide. The morphologies of the peptide nanofiber and the curcumin-encapsulated nanofiber were visualized by transmission electron microscopy. The tumor-targeting activity of the curcumin-encapsulated Nap-GFFYG-RGD peptide nanofiber (f-RGD-Cur) was studied in vitro and in vivo, using Nap-GFFYG-RGE peptide nanofiber (f-RGE-Cur) as the control. Curcumin was encapsulated into the peptide nanofiber, which had a diameter of approximately 10-20 nm. Curcumin showed sustained-release behavior from the nanofibers in vitro. f-RGD-Cur showed much higher cellular uptake in αvß3 integrin-positive HepG2 liver carcinoma cells than did non-targeted f-RGE-Cur, thereby leading to significantly higher cytotoxicity. Ex vivo studies further demonstrated that curcumin could accumulate markedly in mouse tumors after administration of f-RGD-Cur via the tail vein. These results indicate that Nap-GFFYG-RGD peptide self-assembled nanofibers are a promising hydrophobic drug delivery system for targeted treatment of cancer.

[Radio-labeling of T7 peptide with 99mTc and its biodistribution in nude mice bearing non-small cell lung cancer].

BACKGROUND: Lung cancer is a malignant tumor with high mortality rates. This study aims to develop potential candidates of integrin αvß3 imaging agents, which can facilitate the diagnosis and treatment of lung cancer. METHODS: The T7 peptide was labeled with carbonyl technetium. The thin layer chromatography with acetone as the development system was performed to investigate the purity and stability of (99m)Tc-T7. The binding affinity of (99m)Tc-T7 with NCI-H157 tumor cells was determined. The biodistribution of (99m)Tc-T7 in nude mice bearing non-small cell lung carcinoma was observed after injection of (99m)Tc-T7 at 0.5 h, 1 h, 2 h, 4 h, and 8 h, and the radioactive ratio of tumor (T) and non-tumor tissues (NT) was calculated. RESULTS: 99mTc labeled T7 had high radiochemical purity of more than 90%, which does not require further purification, with good stability in vitro. The association and dissociation constant (KD) of (99m)Tc-T7 with NCI-H157 tumor cells was 196.1 nM. (99m)Tc-T7 was mainly metabolism through the internal organs with rapid blood removal. Moreover, the uptake in tumor tissue was significantly higher than the muscle with tumor/muscle ratio of 5.8. In addition, the (99m)Tc-T7 exhibited a transient accumulation in the lungs. CONCLUSIONS: The (99m)Tc-T7 could be prepared using a simple method, had high labeling rate and good stability, and could be accumulated at tumor site. Thus, (99m)Tc-T7 is a potential lung cancer SPECT/CT imaging agent.