Weifeng Ding

Serum APRIL, a potential tumor marker in pancreatic cancer

Abstract Background: A proliferation-inducing ligand (APRIL) is a newly-found member in the tumor necrosis factor (TNF) superfamily. Our previous studies have already confirmed that APRIL is overexpressed in pancreatic cancer tumors, however, it is not expressed or has a weak expression in normal pancreatic gland tissues. Furthermore, there is no report on serum APRIL in patients with pancreatic diseases. Herein, in order to explore the clinical implication of serum APRIL in patients with pancreatic cancer, serum APRIL, together with carcinoembryonic antigen (CEA) and carbohydrate antigen (CA)19-9, was examined. Methods: Serum APRIL was tested by ELISA in patients with pancreatic cancer. Meanwhile, two other conventional serum tumor markers, CEA and CA19-9, were measured by Elecsys 2010 Chemistry Analyzer. Results: Serum APRIL increased in patients with pancreatic cancer, which proved a positive correlation with CEA and CA19-9. When the diagnosis of benign or malignant condition was examined by one tumor marker, the sensitivity of APRIL alone (70.1%) was greater than that of CEA alone (56.7%), and the specificity of APRIL alone (85.5%) was higher than that of CA19-9 alone (83.6%). When examined by a combination of two markers, the sensitivity of the combination of APRIL and CA19-9 was the highest (88.1%), as it was compared with that of APRIL alone, CEA alone and APRIL+CEA, p<0.05. In addition, serum APRIL also correlated with the tumor stage and postoperative survival in patients with pancreatic cancer. Conclusions: Our results indicate that serum APRIL, as a potential biomarker, has a positive diagnosis and prognosis value for pancreatic cancer. Moreover, the combination assay of APRIL and CA19-9 is highly sensitive to pancreatic cancer.

A PROLIFERATION-INDUCING LIGAND: A NEW BIOMARKER FOR NON-SMALL CELL LUNG CANCER

To identify a proliferation-inducing ligand (APRIL) expression profile in tumor tissue and sputum of lung cancer, and evaluate the possibility of an assistant diagnosis of lung cancer by real-time fluorescence quantitative polymerase chain reaction (RTQ-PCR) in sputum as well, the authors analyzed the expression of APRIL mRNA in 75 tissue samples and 71 corresponding sputum samples of lung cancer by RTQ-PCR and analyzed their correlation. APRIL protein expression was also observed in tumor tissues by Western blot and immunohistochemistry. The expression analysis revealed APRIL expression was elevated in non-small cell lung cancer (NSCLC) and the expression of APRIL protein was located in the membrane and cytoplasm of tumor cells by immunohistochemiscal staining. Compared to benign pulmonary disease and healthy volunteers, the expression of APRIL mRNA in sputum of lung cancer was elevated (both P <. 001). When cut-off values for positivity were set at the mean + 2SD of mRNA expression in healthy volunteers, the positive rate for APRIL mRNA expression was 81.7% (58/71) in sputum samples of lung cancer, 3.2% (2/62) in benign pulmonary disease, and 1.5% (1/65) in healthy volunteers. The correlation was evident between the expression level of APRIL mRNA of tissue samples and that of sputum samples (P <. 001, r =. 702). These results support the possibility that the APRIL gene may play a key role in lung cancer, especially in NSCLC. The elevated expression level of APRIL mRNA in sputum of NSCLC suggested that APRIL mRNA may serve as an effective and convenient diagnostic biomarker for NSCLC.

APRIL knockdown suppresses migration and invasion of human colon carcinoma cells

OBJECTIVE The purpose of this study was to investigate the function of a proliferation-inducing ligand (APRIL) gene among the metastatic colorectal cancer (CRC) disease. METHODS Cell adhesion, cell migration and invasion behavior were detected after knockdown of APRIL expression in the colon carcinoma cell line, SW480 by RNAi and the rescue experiments were performed with recombinant human APRIL (rhAPRIL) in vitro. RESULTS This original study indicated that knockdown of APRIL expression strongly inhibited colon carcinoma cell adhesion, cell migration and invasion in vitro. By contrast, reconstitution of APRIL expression with rhAPRIL in these APRILi cells, prominently restores CRC cell migration and invasion. CONCLUSION These results strongly suggest that APRIL play an important role in the tumor development of the invasive or migratory behavior of CRC cells and may be a useful therapeutic target in the malignant colon carcinomas.

Targeting of colorectal cancer growth, metastasis, and anti-apoptosis in BALB/c nude mice via APRIL siRNA

A proliferation-inducing ligand (APRIL) is overexpressed in most tumor cells and tissues, especially in tumors of the alimentary system, such as colorectal cancer (CRC), gastric cancer, and liver cancer. RNA interference (RNAi) has been proved to be a powerful tool for gene knockdown and holds great promise for the treatment of cancer. In this study, the efficacy of RNAi targeting APRIL was analyzed via relevant experiments on human CRC xenografted in BALB/c nude mice. Both the mRNA and protein levels of APRIL were examined after intratumoral injection of APRIL small interfering RNA (siRNA). Meanwhile, pathological tools were utilized to observe the alterations on the aspects of proliferation, metastasis, apoptosis and cellular necrosis by means of detecting proliferating cell nuclear antigen, Ki-67, MMP-2, MMP-9, TIMP-3, TIMP-4, Bcl-2, Bax and Bcl-xL of CRC. In addition, terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labeling (TUNEL) and hematoxylin and eosin staining were also conducted to examine cell apoptosis and necrosis. It was found that grafted human colorectal tumor growth and metastasis were obviously inhibited while tumor cell apoptosis and necrosis were induced after in vivo APRIL siRNA injection into nude mice. The data indicated that silencing of the APRIL gene using RNAi may serve as a novel therapeutic strategy for treatment of CRC.

A novel local anti-colorectal cancer drug delivery system: negative lipidoid nanoparticles with a passive target via a size-dependent pattern

The nontoxic, targeted and effective delivery of nucleic acid drugs remains an important challenge for clinical development. Here, we describe a novel negative lipidoid nanoparticle delivery system, providing entrapment-based transfection agents for local delivery of siRNA to the colorectal cancer focus. The delivery system was synthesized with lipidoid material 98N12-5(1), mPEG2000-C12/C14 glyceride and cholesterol at a desired molar ratio to realize the anionic surface charge of particles, which could alleviate to a larger degree the inflammatory response and immune stimulation of the organism, embodying dramatic biocompatibility. In particular, mPEG2000-C12/C14 glyceride was selected to ameliorate the stability of the delivery system and protection of nucleic acids by extending the tail length of the carbons, crucial also to neutralize the positive charge of 98N12-5(1) to form a resultant anionic particle. In vivo experiments revealed that a particle size of 90 nm perfectly realized a passive target in a size-dependent manner and did not affect the function of the liver and kidneys by a local delivery method, enema. We clarified that the uptake of negative lipidoid nanoparticles internalized through a lipid raft endocytotic pathway with low cytotoxicity, strong biocompatibility and high efficacy. This study suggests that negative lipidoid nanoparticles with enema delivery constitute, uniquely and appropriately, a local anti-colorectal cancer nucleic acid drug delivery platform, and the application of similar modes may be feasible in other therapeutic settings.

Reduced APRIL Expression Induces Cellular Senescence via a HSPG-Dependent Pathway

APRIL, a member of the TNF superfamily, can induce cell proliferation and is overexpressed in most tumor tissues or cells. Nevertheless, it is still unknown about the effect of decreased levels of APRIL expression on tumor cells. In this study, we analyzed APRIL and HSPG expression in the colon carcinoma cell line, SW480 by Western blot and RT-PCR. And the up-regulation of APRIL and HSPG expression was found in SW480. We also observed that knockdown of APRIL levels in SW480, prominently reversed cell proliferation and partially resulted in senescence phenotypes. Furthermore, cellular senescence due to a decreased level of APRIL expression was associated with engagement of HSPG. Thus, our results suggest that low levels of APRIL play an essential role in cellular senescence via a HSPG-dependent signaling pathway in SW480.

APRIL depletion induces cell cycle arrest and apoptosis through blocking TGF-β1/ERK signaling pathway in human colorectal cancer cells

It is well documented that a proliferation-inducing ligand (APRIL), a newly found member of tumor necrosis factor superfamily, overexpressed in the majority of malignancies, plays a potential role in the occurrence and development of these tumors. Herein, we demonstrated that APRIL depletion by using RNA interference in human colorectal cancer (CRC) COLO 205 and SW480 cells resulted in cell proliferation inhibition and evoked cell cycle arrest in G0/G1 phase and apoptosis, coupled with decrease in CDK2, Cyclin D1, Bcl-2 expression and an increase of p21 and Bax expression. In addition, the decreased expression of transforming growth factor-β1 (TGF-β1) and p-ERK was also showed in siRNA-APRIL transfected COLO 205 and SW480 cells, whereas the protein expression levels of Smad2/3, p-Smad2/3, and ERK were not significantly changed. Taken together, our results indicate that APRIL depletion induces cell cycle arrest and apoptosis partly through blocking noncanonical TGF-β1/ERK, rather than canonical TGF-β1/Smad2/3, signaling pathway in CRC cells. Moreover, our study highlights APRIL as a potential molecular target for the therapy of CRC.

Serum sAPRIL: a potential tumor-associated biomarker to colorectal cancer.

OBJECTIVE The purpose of our study was to investigate the serum levels of soluble a-proliferation-inducing ligand (sAPRIL) in patients with colorectal cancer (CRC), benign intestinal disease and healthy volunteers and explore the potential possibility of sAPRIL severing as a CRC biomarker. METHODS Our investigation was conducted on 35 blood samples obtained from CRC patients, 32 blood samples from patients with benign intestinal diseases and 31 blood samples from healthy volunteers. The sAPRIL concentrations were examined by an enzyme-linked immunosorbent assay (ELISA) and data were analyzed with non-parametric Mann-Whitney U-test and X²-test. The correlation relationship between sAPRIL and CEA, as well as sAPRIL and CA19-9 was assessed by non-parametric Spearmens correlation test, respectively. RESULTS The median value of sAPRIL in the malignant group was 10.43 ng/mL, compared with those of the benign group (4.89 ng/mL) and control group (3.30 ng/mL), respectively, which had an obvious significance (P<0.0003). Area under the receiver-operating characteristic (ROC) curve for sAPRIL was 0.854 (95% CI, 0.776-0.933). The optimal cut-off level of sAPRIL was 5.49 ng/mL. Serum sAPRIL had a positive correlation with CEA (r=0.637, P=0.000) and CA19-9 (r=0.357, P=0.008) in 35 patients with colorectal cancer. sAPRIL showed higher sensitivity (82.9%) than those of CEA (74.3%) and CA19-9 (65.7%) in CRC, respectively. CONCLUSION The results indicated that serum sAPRIL, as a potential biomarker, had a positive diagnostic value for colorectal cancer.

Amelioration of Colorectal Cancer Using Negative Lipidoid Nanoparticles to Encapsulate siRNA Against APRIL by Enema Delivery Mode

A proliferation-inducing ligand (APRIL) is a key cell proliferation-regulatory molecule and have been investigated well enough in immunity regulation and a few of immune diseases. APRIL can stimulate tumor cell growth and is up-expressed in cancer tissues, especially in CRC (colorectal cancer). However, whether inhibition of APRIL can regulate tumor-relative genes expression in vivo and subsequently ameliorate the pathological progress of CRC remains obscure. To address this question, we developed a novel negative lipidoid nanoparticles (NLNs) encapsulating small interference RNA (siRNA) for selectively silencing APRIL in the parenchyma of CRC focus in vivo, which uptake proceeded through a lipid raft endocytotic pathway. Local enema delivery of APRIL-NLNs silenced APRIL in CRC cells and animal models, and then ameliorated experimentally the progress of CRC by suppressing CRC cell proliferation, metastasis, and apoptosis-related cytokine expression and did not affect the function of liver and kidneys and not trigger the immune response of CRC models. This study reveals APRIL to be a potential anti-CRC target by in vivo experiments, and suggests that the application of similar modes of siRNA delivery may be feasible in other therapeutic settings.