The tumour neovasculature-homing dimeric peptide GX1 demonstrates antiangiogenic activity in the retinal neovasculature.

Identification of molecules specific to the retinal neovasculature will promote antiangiogenic therapy with enhanced targeting ability. The specificity of phage-displayed peptide GX1 (a cyclic 7-mer peptide motif CGNSNPKSC) to gastric cancer neovasculature has been extensively confirmed both in vitro and in vivo. To investigate the potential application of GX1 in antiangiogenic therapy targeting retinal angiogenesis-related diseases, we performed immunohistochemistry and immunofluorescence analyses. GX1 demonstrated positive staining in the retinal neovasculature in an oxygen-induced mouse model of retinopathy (OIR) as well as in rat retinal microvasculature endothelial cells (RMECs), confirming the major role of the GX1 receptor during retinal angiogenesis. Dimeric GX1 was synthesized to increase the binding affinity to the GX1 receptor, and the antiangiogenic effects were examined in RMECs in vitro and the retinal neovasculature in the OIR in vivo. Cell proliferation was evaluated using a Cell Counting Kit-8 (CCK-8) assay, revealing that compared with the GX1 monomer, dimeric GX1 significantly inhibited RMEC proliferation (P < 0.05). This finding may be attributed to the enhanced (P < 0.05) apoptosis induced by dimeric GX1 in RMECs based on results obtained from TUNEL, flow cytometric and cell cycle analyses. In RMECs, in vitro cell migration and tube formation were significantly inhibited following exposure to dimeric GX1. Intravitreal administration of dimeric GX1 resulted in a greater reduction in the retinal neovascularization in vivo than administration of the GX1 monomer (P < 0.05). In conclusion, dimeric GX1 showed greater inhibition of angiogenesis than monomeric GX1 and could be a promising agent for antiangiogenic therapy in retinal angiogenesis-related diseases.

68Ga-Labeled GX1 Dimer: A Novel Probe for PET/Cerenkov Imaging Targeting Gastric Cancer.

PURPOSE: To synthesize the dimer of GX1 and identify whether its affinity and targeting are better than those of GX1. To prepare 68Ga-DOTA-KEK-(GX1)2 and to apply it to PET and Cerenkov imaging of gastric cancer. METHODS: 68Ga-DOTA-KEK-(GX1)2 was prepared, and the labeling yield and stability were determined. Its specificity and affinity were verified using an in vitro cell binding assay and competitive inhibition test, cell immunofluorescence, and cell uptake and efflux study. Its tumor-targeting ability was determined by nano PET/CT and Cerenkov imaging, standardized uptake value (SUV), signal-to-background ratio (SBR) quantification, and a biodistribution study in tumor-bearing nude mice. RESULTS: 68Ga-DOTA-KEK-(GX1)2 was successfully prepared, and the labeling yield was more than 97%. It existed stably for 90 min in serum. The binding of 68Ga-DOTA-KEK-(GX1)2 to cocultured HUVECs (Co-HUVECs) was higher than that to human umbilical vein endothelial cells (HUVECs), BGC823 cells, and GES cells. It was also higher than that of 68Ga-DOTA-GX1, indicating that the dimer did improve the specificity and affinity of GX1. The binding of KEK-(GX1)2 to Co-HUVECs was significantly higher than that of GX1. Additionally, the uptake of 68Ga-DOTA-KEK-(GX1)2 by Co-HUVECs was higher than that of 68Ga-DOTA-GX1 and reached a maximum at 60 min. Nano PET/CT and Cerenkov imaging showed that the tumor imaging of the nude mice injected with 68Ga-DOTA-KEK-(GX1)2 was clear, and the SUV and SBR value of the tumor sites were significantly higher than those of the nude mice injected with 68Ga-DOTA-GX1, indicating that the probe had better targeting in vivo. Finally, the biodistribution showed quantitatively that when organs such as the kidney and liver metabolized rapidly, the radioactivity of the tumor site of the nude mice injected with 68Ga-DOTA-KEK-(GX1)2 decreased relatively slowly. At the same time, the percentage of injected dose per gram (%ID/g) of the tumor site was higher than that of other normal organs except the liver and kidney at 60 min, which indicated that the tumor had good absorption of the probe. CONCLUSION: GX1 was modified successfully, and the in vivo and in vitro properties of the GX1 dimer were significantly better than those of GX1. The imaging probe, 68Ga-DOTA-KEK-(GX1)2, was successfully prepared, which provides a candidate probe for PET and Cerenkov diagnosis of gastric cancer.

188Re-labeled GX1 dimer as a novel dual-functional probe targeting TGM2 for imaging and antiangiogenic therapy of gastric cancer.

PURPOSE: The GX1 peptide (CGNSNPKSC) can specifically bind to TGM2 and possesses the ability to target the blood vessels of gastric cancer. This study intends to develop an integrated dual-functional probe with higher affinity, specificity and targeting and to characterize it in vivo and in vitro. METHODS: The dimer and tetramer of GX1 were prepared using cross-linked PEG and labeled with 99mTc. The best targeting probe [PEG-(GX1)2] was selected by gamma camera imaging in nude mouse models of gastric cancer. 188Re-PEG-(GX1)2 was prepared and characterized through cell binding analysis and competitive inhibition experiments, gamma camera imaging, MTT analysis and flow cytometry, BLI, immunohistochemistry, HE staining and biochemical analysis. RESULTS: PEG-(GX1)2 bound specifically to Co-HUVEC with higher affinity than GX1. 188Re-PEG-(GX1)2 had better ability to target gastric cancer in tumor-bearing nude mice and higher T/H ratios than 188Re-GX1. 188Re-PEG-(GX1)2 inhibited the growth of Co-HUVEC and induced apoptosis, and its effects were more robust than those of 188Re-GX1. BLI showed that 188Re-PEG-(GX1)2 inhibited tumor proliferation in vivo with a stronger effect than 188Re-GX1. Compared with 188Re-GX1, 188Re-PEG-(GX1)2 suppressed tumor angiogenesis and tumor cell proliferation and induced tumor cell apoptosis in vivo. The 188Re-PEG-(GX1)2 group did not cause visible changes in liver and kidney morphology and function in vivo. CONCLUSION: The dimer of GX1 was synthesized by using cross-linked PEG, and then 188Re-PEG-(GX1)2 was prepared. This radiopharmaceutical played both diagnostic and therapeutic functions, and gamma camera imaging could be utilized to detect the distribution of drugs in vivo during treatment. Through a series of experiments in vitro and in vivo, the feasibility of the drug was confirmed, and these results laid the foundation for the subsequent development and application of GX1.

Kinetic modeling and analysis of dynamic bioluminescence imaging of substrates administered by intraperitoneal injection.

BACKGROUND: Bioluminescence imaging (BLI) has been found to have diverse applications in the life sciences and medical research due to its ease of use and high sensitivity. From kinetics analysis, dynamic imaging studies have significant advantages for diagnosis when compared to traditional static imaging studies. This work focuses on modeling and quantitatively analyzing the dynamic data produced from the intraperitoneal (IP) injection of D-luciferin in longitudinal BLI, aiming to provide a powerful tool for monitoring the growth of tumors. METHODS: We constructed a three-compartment pharmacokinetic (PK) model and employed the standard Michaelis-Menten (M-M) kinetics to investigate the dynamic BLI data produced from the IP injection of D-luciferin. The 3 compartments were the plasma compartment, the non-specific compartment, and the specific compartment. The validity of this PK model was tested by the dynamic BLI data of MKN28M-luc xenograft mice, along with the published longitudinal dynamic BLI data of B16F10-luc xenograft mice. RESULTS: The R-squares between the simulated lines and the measurement were 1 and 0.99, respectively, for the mice data and the published data. In addition, the 2 kinetic macroparameters obtained reflected the rate of tumor growth in vivo. In particular, the values of macroparameters A showed a significant dependence on tumor surface area. CONCLUSIONS: The proposed PK model may be an effective tool for use in drug development programs and for monitoring the response of tumors to treatment.

Clinicopathological and prognostic impact of somatic mutations in Chinese patients with clear cell renal cell carcinoma.

BACKGROUND: The study of the genomic landscape of Chinese clear cell renal cell carcinoma (ccRCC) entered its nascence in recent years, and the clinical relevance of individual genes in Chinese ccRCC has not yet been researched. The study aimed to explore the relationships between somatic mutations and clinical behaviors in Chinese ccRCC. METHODS: Tumor tissue samples were obtained from 105 Chinese patients with ccRCC and deep sequencing targeting 556 cancer genes was performed. Correlation analysis, receiver operator characteristic (ROC) analysis and survival analysis were carried out using SPSS software. RESULTS: A total of 41 genes were used to investigate the relationship between genes and clinical behaviors. We found that different clinical indices were mutually correlated, and there were 12 genes associated with clinical indices. The Kaplan-Meier curves showed that high Fuhrman grade and metastatic disease at diagnosis were significantly associated with poor prognosis. Mutations in BAP1, PTEN, ERBB2, TP53, CDK8, TSC1, SETD2, or SPEN were significantly associated with poor prognosis, consistent with the results of The Cancer Genome Atlas (TCGA) cohort. Mutation of BTG1 occurred much more frequently in Chinese ccRCC (10.5%) than in the TCGA cohort (0.60%), and it was associated with a better prognosis. CONCLUSIONS: A total of 8 genes (BAP1, PTEN, ERBB2, TP53, CDK8, TSC1, SETD2, and SPEN) were found to be associated with poor prognosis of ccRCC, and a new gene (BTG1) was possibly associated with the good prognosis of Chinese ccRCC.

Intravenous Administration-Oriented Pharmacokinetic Model for Dynamic Bioluminescence Imaging.

OBJECTIVE: In vivo bioluminescence imaging (BLI) is a promising tool for monitoring the growth and metastasis of tumors. However, quantitative BLI research based on intravenous (IV) injection is limited, which greatly restricts its further application. To address this problem, we designed a pharmacokinetic (PK) model which is suitable for applying on IV administration of small amounts of D-Luciferin. METHODS: After three weeks of postimplantation, mkn28-luc xenografted mice were subjected to 40-min dynamic BLI immediately following D-Luciferin intravenous injection on days 1, 3, 5, 7, and 9. Furthermore, the PK model was applied on dynamic BLI data to obtain the sum of kinetic rate constants (SKRC). RESULTS: Results showed that the SKRC values decreased rapidly with the growth of the tumor. There was a statistical difference between the SKRC values measured at different time points, while the time point of luminous intensity peak was unaffected by the growth of the tumor. CONCLUSION: In short, our results imply that dynamic BLI combined with our PK model can predict tumor growth earlier and with higher sensitivity compared to the conventional method, which is crucial for improving drug evaluation efficacy. In addition, the dynamic BLI may provide a valuable reference for the noninvasive acquiring arterial input function, which may also provide a new application prospect for hybrid PET-optical imaging.

Overexpression of serum response factor is correlated with poor prognosis in patients with gastric cancer.

Serum response factor (SRF) is highly expressed in many tumors, including gastric cancer. However, the exact prognostic utility of SRF in patients with gastric cancer remains unclear. Therefore, in this study, we investigated the expression and prognostic value of SRF in patients with gastric cancer. SRF expression was detected by immunohistochemistry in 149 gastric cancer samples. The relationship between SRF expression and clinicopathological characteristics along with the prognostic significance of SRF in disease-free survival and overall survival of patients was analyzed. We found that the expression of SRF was significantly increased in gastric cancer tissues compared with adjacent noncancerous tissues. Overexpression of SRF was significantly correlated with histologic differentiation, invasion depth, lymph node metastasis, and TNM stage. Furthermore, disease-free survival rate and overall survival rate were both significantly lower for patients with high SRF expression. Multivariate Cox regression analysis identified high SRF expression as an independent prognostic factor for disease-free survival but not for overall survival. Our findings indicate that overexpression of SRF may play an important role in human gastric cancer recurrence and prognosis. SRF may serve as a predictive marker for prognosis of gastric cancer.

FOXM1 promotes proliferation in human hepatocellular carcinoma cells by transcriptional activation of CCNB1.

The transcription factor Forkhead box protein M1 (FOXM1) plays critical roles in cancer development and progression, including human hepatocellular carcinoma (HCC). However, the regulatory role and underlying mechanisms of FOXM1 is still limited. Here, we found that the high level expression of FOXM1 and CCNB1 is closely associated with poor prognosis in HCC patients. And FOXM1 and CCNB1 were overexpressed concomitantly in liver tumor tissues. Knockdown of FOXM1 significantly inhibited the expression levels of CCNB1 in HCC cell lines at both the mRNA and protein levels. Mechanistic studies revealed that FOXM1 binds directly to the promoter region of CCNB1 and regulates the expression levels of the CCNB1 gene in the transcriptional level. Furthermore, the loss of functional and rescue experiments showed that CCNB1 is essential for FOXM1-driven proliferation in HCC cells. In the present study, our results partially explained the dysregulated expression of FOXM1 play an important role in proliferation of human hepatocellular carcinoma cells via transcriptional activation of CCNB1 expression. And it also highlights a FOXM1/CCNB1 axis could be a potential target for the treatment of HCCs.

Combined use of alcohol in conventional chemical-induced mouse liver cancer model improves the simulation of clinical characteristics of human hepatocellular carcinoma.

Liver cancer is the one of most common types of cancer and the 2nd cause of cancer-associated mortalities worldwide. Establishing appropriate animal models of liver cancer is essential for basic and translational studies. The present study evaluated the effects of the combined use of alcohol with a conventional chemical-induced mouse liver cancer model. The treatment of alcohol/diethylnitrosamine (DEN)/carbon tetrachloride (CCl4) in the mice of experimental groups resulted in a series of pathological changes in the liver. Liver inflammation, fibrosis, cirrhosis and hepatocellular carcinoma were identified, and this method used less time (1-5 months) for inducement compared with the conventional chemical-induced method alone. In addition, murine α-fetoprotein (mAFP) was expressed throughout and ultrastructural features met the criteria for liver cancer. Fatty degeneration of pancreas, reduced blood glucose levels, and increased spleen weight were observed. These results indicated that an AFP-secreting hepatocellular carcinoma model of BALB/c mouse was successfully developed. The disease process and morphological changes met the criterion of the liver cancer process. Therefore the model developed in the present study may be an ideal animal model for studying the occurrence and development of liver cancer.

Loss of Barx1 promotes hepatocellular carcinoma metastasis through up-regulating MGAT5 and MMP9 expression and indicates poor prognosis.

Metastasis is the major dominant reason for poor prognosis of hepatocellular carcinoma (HCC) after surgical treatment. However, the molecular mechanism of metastasis has not been well characterzied. Here, we report a novel function of Barx homeobox1 (Barx1) in inhibiting HCC invasion and metastasis. Barx1 expression is significantly decreased in human HCC tissues than in adjacent non-tumorous tissues and normal liver tissues. Low Barx1 expression is correlated with higher tumor-nodule-metastasis stage and indicates poor prognosis. Down-regulation of Barx1 promotes HCC migration, invasion and metastasis, whereas up-regulation of Barx1 inhibits HCC migration, invasion and metastasis. Mannosyl (alpha-1,6-)-glycoprotein beta-1,6-N-acetyl-glucosaminyltransferase 5 (MGAT5) and matrix metallopeptidase 9 (MMP9) are direct target genes of Barx1. Knockdown of Barx1 up-regulates MGAT5 and MMP9 expression in HCC cells with low metastatic capability, whereas over-expression of Barx1 suppresses their expression in HCC cells with high metastatic capability. Knockdown of both MGAT5 and MMP9 significantly decreases the invasion and metastasis abilities induced by Barx1 knockdown. Barx1 expression is negatively correlated with MGAT5 and MMP9 expression in human HCC tissues. Patients with low expression of Barx1 and high expression of MGAT5 or MMP9 are associated with poorer prognosis. Thus, loss of Barx1 represents a prognostic biomarker in human HCC patients.

Investigation of injection dose and camera integration time on quantifying pharmacokinetics of a Cy5.5-GX1 probe with dynamic fluorescence imaging in vivo.

The aim of this article is to investigate the influence of a tracer injection dose (ID) and camera integration time (IT) on quantifying pharmacokinetics of Cy5.5-GX1 in gastric cancer BGC-823 cell xenografted mice. Based on three factors, including whether or not to inject free GX1, the ID of Cy5.5-GX1, and the camera IT, 32 mice were randomly divided into eight groups and received 60-min dynamic fluorescence imaging. Gurfinkel exponential model (GEXPM) and Lammertsma simplified reference tissue model (SRTM) combined with a singular value decomposition analysis were used to quantitatively analyze the acquired dynamic fluorescent images. The binding potential (Bp) and the sum of the pharmacokinetic rate constants (SKRC) of Cy5.5-GX1 were determined by the SRTM and EXPM, respectively. In the tumor region, the SKRC value exhibited an obvious trend with change in the tracer ID, but the Bp value was not sensitive to it. Both the Bp and SKRC values were independent of the camera IT. In addition, the ratio of the tumor-to-muscle region was correlated with the camera IT but was independent of the tracer ID. Dynamic fluorescence imaging in conjunction with a kinetic analysis may provide more quantitative information than static fluorescence imaging, especially for a priori information on the optimal ID of targeted probes for individual therapy.

In vivo quantifying molecular specificity of Cy5.5-labeled cyclic 9-mer peptide probe with dynamic fluorescence imaging.

We quantified molecular specificity of Cy5.5-GX1 in vivo with dynamic fluorescence imaging to better understand its kinetic properties. According to whether or not free GX1 was injected and when it was injected, twelve of BGC-823 xenografted mice were randomly divided into three groups and underwent a 60 minute dynamic fluorescence scanning. Combined with a principal-component analysis, the binding potential (Bp) of the probe was determined by both Logan graphical analysis with reference tissue model (GARTM) and Lammertsma simplified reference tissue model (SRTM). The sum of the pharmacokinetic rate constants (SKRC) was quantified by the Gurfinkel exponential model (GEXPM). Cy5.5-GX1 specifically targeted tumor both in vitro and in vivo. We obtained similar quantification results of Bp (GARTM Bp = 0.582 ± 0.2655, SRTM Bp = 0.618 ± 0.2923), and obtained a good linear relation between the Bp value and the SKRC value. Our results indicate that the SKRC value is more suitable for an early-stage kinetic data analysis, and the Bp value depicts kinetic characteristics under the equilibrium state. Dynamic fluorescence imaging in conjunction with various kinetic models are optimal tools to quantify molecular specificity of the Cy5.5-GX1 probe in vivo.

POU2F2-oriented network promotes human gastric cancer metastasis.

BACKGROUND AND AIMS: Aberrant upregulation of POU2F2 expression has been discovered in metastatic gastric cancer (GC). However, the mechanisms underlying the aberrant upregulation and the potential functions of POU2F2 remain uncertain. DESIGN: The role and mechanism of POU2F2 in GC metastasis were investigated in gastric epithelial cells, GC cell lines and an experimental metastasis animal model by gain of function and loss of function. Upstream and downstream targets of POU2F2 were selected by bioinformatics and identified by luciferase reporter assay, electrophoretic mobility shift assay and chromatin immunoprecipitation PCR. The influence of miR-218 on its putative target genes (POU2F2, ROBO1 and IKK-ß) and GC metastasis was further explored via in vitro and in vivo approaches. RESULTS: Increased POU2F2 expression was detected in metastatic GC cell lines and patient samples. POU2F2 was induced by the activation of nuclear factor (NF)-κB and, in turn, regulated ROBO1 transcription, thus functionally contributing to GC metastasis. Finally, miR-218 was found to suppress GC metastasis by simultaneously mediating multiple molecules in the POU2F2-oriented network. CONCLUSIONS: This study demonstrated that NF-κB and the SLIT2/ROBO1 interaction network with POU2F2 as the central part may exert critical effects on tumour metastasis. Blocking the activation of the POU2F2-oriented metastasis network using miR-218 precursors exemplified a promising approach that sheds light on new strategies for GC treatment.

Targeted delivery of CXCR4-siRNA by scFv for HER2(+) breast cancer therapy.

Therapeutics based on short interfering RNAs (siRNAs) have great potential to treat human diseases. However, the clinical application of siRNAs has been limited by their poor intracellular uptake, low serum stability, and inability to target specific cells. In this study, we addressed this lack of specificity by synthesizing a molecularly targeted CXCR4-siRNA (CXCR4si) for the treatment of HER2(+) breast cancers using a HER2-scFv-arginine nonamer peptide fusion protein (e23sFv-9R) as an siRNA carrier. The e23sFv-9R binding siRNA is able to specifically deliver the siRNA to HER2(+) breast cancer cells and concentrate and persist in orthotopic HER2(+) breast cancer xenografts for at least 36 h. CXCR4si delivered by e23sFv-9R inhibited CXCR4 gene expression, reduced proliferation and metastasis and induced apoptosis in the HER2(+) breast cancer BT-474 cell line in vitro. Moreover, the systemic delivery of CXCR4si by e23sFv-9R is able to suppress tumor growth, reduce metastasis and prolong survival in mice bearing HER2(+) xenografts. This approach causes no systemic toxicity and does not activate the innate immune response, suggesting that a fusion protein carrying CXCR4si shows promise in the treatment of HER2-overexpressing breast cancer.

Antidepressant-like effects of magnesium lithospermate B in a rat model of chronic unpredictable stress.

CONTEXT: Magnesium lithospermate B (MLB), an active polyphenol acid of Danshen [Radix Salviae miltiorrhizae (Labiatae)], shows neuroprotective and anti-inflammatory effects in vivo and in vitro. OBJECTIVE: We hypothesized that MLB might exert antidepressant-like effects by targeting the neuroinflammatory signals. MATERIALS AND METHODS: Sprague-Dawley rats were subjected to the chronic unpredictable stress (CUS) protocol. Rats in the control group received no CUS during the whole experiment. In the model group, rats were exposed to CUS for 7 weeks. From the beginning of the 5th week, model group rats were randomly grouped and subjected to different treatments. In the experiment, control and model group rats were intraperitoneally (i.p.) injected with saline. MLB was dissolved in saline to give a final concentration, and the rats were injected (i.p.) with 15, 30, or 60 mg/kg MLB once a day for 3 weeks. RESULTS: MLB administration significantly reduced: (1) the immobility time in the forced swimming test (19 s, p < 0.05); (2) the immobility time in the tail suspension test (76.3 s, p < 0.05); (3) the corticosterone (CORT) concentrations in the serum (21.7 nmol/L, p > 0.05); (4) the pro-inflammatory cytokine levels in the serum - TNF-α (92.1 pg/ml, p < 0.05), IL-1ß (86.9 pg/ml, p < 0.05), and IL-6 (93.8 pg/ml, p < 0.05); (5) pro-inflammatory cytokine levels in tissue - TNF-α (3.2 pg/mg protein, p < 0.05), IL-1ß (1.5 pg/mg protein, p > 0.05), and IL-6 (6.3 pg/mg protein, p < 0.05); and (6) phospho-NF-κB (1.6, p < 0.05) and phospho-IκB-α (0.4, p < 0.05) expression in tissue. DISCUSSION AND CONCLUSION: The results suggested that MLB might exert therapeutic actions on depression-like behavior and the HPA axis hyperactivity in CUS rats, and the mechanisms underlying the antidepressant-like effects of MLB might be mediated by regulation of the expression of NF-κB and IκB-α in rats.

Evaluation of Tc-99 m Labeled Dimeric GX1 Peptides for Imaging of Colorectal Cancer Vasculature.

PURPOSE: This study aimed to evaluate the potential of PEGylated dimeric GX1 peptide as a radiotracer for imaging of colorectal cancer vasculature in a LoVo tumor xenografted mouse model. PROCEDURES: The [(99m)Tc]PEG-(GX1)2 peptide was synthesized and identified. Confocal immunofluorescence analysis, receptor binding assay, and competitive inhibition assay were performed to evaluate the binding specificity and the receptor binding affinity of PEG-(GX1)2 to Co-human umbilical vein endothelial cells (HUVECs). Single photon emission computed tomography imaging and biodistribution were performed to evaluate the targeting ability of PEG-(GX1)2 to colorectal cancer. RESULTS: The studies in vitro suggested that PEG-(GX1)2 co-localized with Factor VIII in the perinuclear cytoplasm of Co-HUVECs and bound specifically to Co-HUVECs with a high affinity. The studies in vivo demonstrated that the targeting efficacy of PEG-(GX1)2 was superior to GX1. CONCLUSIONS: PEGylation improved the affinity and the targeting ability of the GX1 peptide. PEG-(GX1)2 is a more promising probe for imaging of colorectal vasculature than GX1.

Evaluation of 68Ga-labeled MG7 antibody: a targeted probe for PET/CT imaging of gastric cancer.

MG7-Ag, a specific gastric cancer-associated antigen, can be used to non-invasively monitor gastric cancer by molecular imaging with positron emission tomography/computed tomography (PET/CT). In this study, we prepared and evaluated a (68)Ga-labeled MG7 antibody as a molecular probe for nanoPET/CT imaging of gastric cancer in a BGC-823 tumor xenografted mouse model. Macrocyclic chelator 1,4,7-triazacyclononane-N,N0,N00-triacetic acid (NOTA)-conjugated MG7 antibody was synthesized and radiolabeled with (68)Ga (t1/2 = 67.71 min). Then, (68)Ga-NOTA-MG7 was tested using in vitro cytological studies, in vivo nanoPET/CT and Cerenkov imaging studies as well as ex vivo biodistribution and histology studies. The in vitro experiments demonstrated that (68)Ga-NOTA-MG7 has an excellent radiolabeling efficiency of approximately 99% without purification, and it is stable in serum after 120 min of incubation. Cell uptake and retention studies confirmed that (68)Ga-NOTA-MG7 has good binding affinity and tumor cell retention. For the nanoPET imaging study, the predominant uptake of (68)Ga-NOTA-MG7 was visualized in tumor, liver and kidneys. The tumor uptake reached at its peak (2.53 ± 0.28%ID/g) at 60 min pi. Cherenkov imaging also confirmed the specificity of tumor uptake. Moreover, the biodistribution results were consistent with the quantification data of nanoPET/CT imaging. Histologic analysis also demonstrated specific staining of BGC-823 tumor cell lines.

In vivo molecular imaging of gastric cancer in human-murine xenograft models with confocal laser endomicroscopy using a tumor vascular homing peptide.

The early detection of premalignant lesions and cancers are very important for improving the survival of patients with gastric malignancies. Confocal laser endomicroscopy (CLE) is a novel imaging tool for achieving real-time microscopy during the ongoing endoscopy at subcellular resolution. In the present study, to evaluate the feasibility of real-time molecular imaging of GEBP11 by CLE in gastric cancer, CLE was performed on two types of tumor-bearing mice models, as well as surgical specimens of patients with gastric cancer, after the application of GEBP11. A whole-body fluorescent imaging device was first used to screen for the strongest specific fluorescent signal in xenograft models. Next, the tumor sites, as well as human tissues, were scanned with CLE. After this, targeted specimens were obtained for fluorescence microscopy and histology. We confirmed that GEBP11 could specifically bind to co-HUVECs by means of CLE in cell experiments. Thereafter, a specific signal was observed in both subcutaneous and orthotopic xenograft models in vivo after the injection of FITC-GEBP11 via tail vein, whereas the group injected with FITC-URP showed no fluorescent signals. In human tissues, a specific signal of GEBP11 was observed in 26/28 neoplastic specimens and in 8/28 samples of non-neoplastic specimens from the patients (p < 0.01). The findings from ex vivo immunofluorescence microscopy of cryostat sections correlated well with that obtained by CLE. These findings indicate that the peptide, GEBP11, might be a potential candidate for the molecular imaging of gastric cancer.

GX1 targeting delivery of rmhTNFα evaluated using multimodality imaging.

GX1 is a tumor targeting peptide. In this study, we evaluated the antitumor efficacy of a GX1-derived fusion toxin, GX1-rmhTNFα, and investigated its targeting efficiency and pharmacokinetics in vivo using multimodality imaging. Flow cytometry revealed a greater level of cell apoptosis induced by GX1-rmhTNFα (27.1%) compared with rmhTNFα or a saline control (13.7% and 4.7%, respectively). SPECT (single-photon emission computed tomography) demonstrated high accumulation of GX1-rmhTNFα in tumor site. Biodistribution studies indicated GX1-rmhTNFα was cleared by the liver and kidney, and the drug may not cross the blood-brain barrier. In addition, bioluminescence imaging (BLI) showed that GX1-rmhTNFα caused a satisfactory delay in tumor growth in both subcutaneous and orthotopic cancer models. Contrast-enhanced ultrasound (CEUS) and CD31 staining revealed a loss in blood perfusion and vasculature. TUNEL and Ki67 staining validated the in vivo results. Biochemical analyses revealed limited renal and hepatic toxicity of GX1-rmhTNFα. This study demonstrated that GX1-rmhTNFα is a safe and potent anticancer agent that may have great potential for the targeted therapy of gastric cancer.

Magnesium lithospermate B improves myocardial function and prevents simulated ischemia/reperfusion injury-induced H9c2 cardiomyocytes apoptosis through Akt-dependent pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Magnesium lithospermate B (MLB), an active polyphenol acid of Radix Salviae Miltiorrhizae (Danshen), showed a wide range of pharmacological activities in cardiovascular diseases. However, its role in protection against ischemia/reperfusion injury in H9c2 cardiomyocytes has not been elucidated. This study was aimed to investigate the protective effect and potential molecular mechanisms of MLB on apoptosis in H9c2 cardiomyocytes in vitro. MATERIALS AND METHODS: We tested cell viability, shortening amplitude, necrosis, apoptosis, and the expression levels of Akt, phosphorylated Akt, Bcl-2 and Bax after 2-h simulated ischemia and 24-h simulated reperfusion in H9c2 cardiomyocytes. We further observed the contractile function in hearts after they were subjected to global 30-min ischemia and 180-min reperfusion. RESULTS: Pretreatment with MLB markedly increased cell viability and while reducing evidence of necrosis and apoptosis in H9c2 cardiomyocytes. In addition, the expression of Bcl-2 and Bax protein was modulated. The results also showed that MLB significantly increased phosphorylation of Akt and that this phosphorylation can be partially inhibited by phosphoinositide 3-kinase/Akt inhibitor. Furthermore, MLB improved MI/R-induced myocardial contractile function. CONCLUSION: Our results showed that MLB prevents I/R-induced myocardial damage by reducing necrosis and apoptosis in H9c2 cardiomyocytes and improving myocardial function in rat hearts.