Zhenyu Ji

The membrane-cytoskeletal protein 4.1N is involved in the process of cell adhesion, migration and invasion of breast cancer cells

Protein 4.1N belongs to the protein 4.1 superfamily that links transmembrane proteins to the actin cytoskeleton. Recent evidence has shown that protein 4.1 is important in tumor suppression. However, the functions of 4.1N in the metastasis of breast cancer are largely unknown. In the present study, MCF-7, T-47D and MDA-MB-231 breast cancer cell lines with various metastatic abilities were employed. Protein 4.1N was found to be expressed in poorly metastatic MCF-7 and middle metastatic T-47D cell lines, and was predominantly associated with cell-cell junctions. However, no 4.1N expression was detected in the highly metastatic MDA-MB-231 cells. Moreover, re-expression of 4.1N in MDA-MB-231 cells inhibited cell adhesion, migration and invasion. The results suggest that protein 4.1N is a negative regulator of cell metastasis in breast cancer.

A Recombinant DNA Plasmid Encoding the sIL-4R-NAP Fusion Protein Suppress Airway Inflammation in an OVA-Induced Mouse Model of Asthma

Asthma is a chronic inflammatory airway disease. It was prevalently perceived that Th2 cells played the crucial role in asthma pathogenesis, which has been identified as the important target for anti-asthma therapy. The soluble IL-4 receptor (sIL-4R), which is the decoy receptor for Th2 cytokine IL-4, has been reported to be effective in treating asthma in phase I/II clinical trail. To develop more efficacious anti-asthma agent, we attempt to test whether the Helicobacter pylori neutrophil-activating protein (HP-NAP), a novel TLR2 agonist, would enhance the efficacy of sIL-4R in anti-asthma therapy. In our work, we constructed a pcDNA3.1-sIL-4R-NAP plasmid, named PSN, encoding fusion protein of murine sIL-4R and HP-NAP. PSN significantly inhibited airway inflammation, decreased the serum OVA-specific IgE levels and remodeled the Th1/Th2 balance. Notably, PSN is more effective on anti-asthma therapy comparing with plasmid only expressing sIL-4R.

Antitumor and immunomodulatory effects of recombinant fusion protein rMBP-NAP through TLR-2 dependent mechanism in tumor bearing mice.

The pro-inflammatory and immunomodulatory properties of Helicobacter pylori neutrophil activating protein (Hp-NAP) not only make it to play an important role in disease pathogenesis but also make it to be a potential candidate for therapeutic applications, including vaccine and drug development. Our previous work demonstrated that the recombinant Hp-NAP fused with the maltose binding protein of Escherichia coli (rMBP-NAP) play an important role in regulating the differentiation of Th1 cells. In this study, we investigated the ability of rMBP-NAP to induce antitumor immunity using two murine models of hepatoma H22 and sarcoma S180. Subcutaneous administration of mice with rMBP-NAP resulted in an about 40%-50% decrease of tumor growth compared with that of the control mice. Splenocytes from the tumor-bearing mice treated with rMBP-NAP showed a significant accumulation of CD4(+) IFN-γ-secreting cells, which is a cytokine profile of Th1 cells. Furthermore, intravenous injection of T2.5, toll like receptor (TLR) 2 blocking antibody, significantly recede the antitumor effect of rMBP-NAP and the production of IFN-γ induced by rMBP-NAP. Our findings indicate that potentiality of rMBP-NAP to be a candidate for the development of immunomodulatory antitumoral drugs.

Protein 4.1R is Involved in the Transport of 5‐Aminolevulinic Acid by Interaction with GATs in MEF Cells

5‐aminolevulinic acid (5‐ALA)‐based photodynamic therapy (PDT) has been successfully used in the treatment of cancers. However, the mechanism of 5‐ALA transportation into cancer cells is still not fully elucidated. Previous studies have confirmed that the efficiency of 5‐ALA‐PDT could be affected by the membrane skeleton protein 4.1R. In this study, we investigated the role of 4.1R in the transport of 5‐ALA into cells. Wild‐type (4.1R+/+) and 4.1R gene knockout (4.1R−/−) mouse embryonic fibroblast (MEF) cells were incubated with 1 mm 5‐ALA and different concentrations of specific inhibitors of GABA transporters GAT (1‐3). Our results showed that the inhibition of GAT1 and GAT2 in particular markedly attenuated the intracellular PpIX production, reactive oxygen species (ROS) level and 5‐ALA‐induced photodamage. However, the inhibition of GAT3 did not show such effects. Further research showed that 4.1R−/− MEF cells had a lower expression of GAT1 and GAT2 than 4.1R+/+ MEF cells. Additionally, 4.1R directly bound to GAT1 and GAT2. Taken together, GAT1 and GAT2 transporters are involved in the uptake of 5‐ALA in MEF cells. 4.1R plays an important role in transporting 5‐ALA into cells via at least partly interaction with GAT1 and GAT2 transporters in 5‐ALA‐PDT.

Toll‑like receptor agonist rMBP‑NAP enhances antitumor cytokines production and CTL activity of peripheral blood mononuclear cells from patients with lung cancer

Toll-like receptor (TLR) agonists are known for their ability to inhibit tumor progression via enhancing antitumor cytokines production and cytotoxic T lymphocyte (CTL) activity. Recombinant Helicobacter pylori neutrophil-activating protein fused with maltose-binding protein (rMBP-NAP) has been reported as a novel TLR agonist for antitumor treatment in murine models. The present study aimed to determine the potential and efficacy of the rMBP-NAP for antitumor treatment prior to further clinical trials. The rMBP-NAP was expressed and purified for subsequent experiments. Peripheral blood mononuclear cells (PBMCs) from health donors and patients with lung cancer (LC) were incubated with PBS and 0.2 mg/ml rMBP-NAP. Antitumor cytokines production was assayed using ELISA and reverse transcription-quantitative polymerase chain reaction analysis. The cytolytic activity of PBMCs and the number of Interferon-γ (IFN-γ)-secreting cells were assayed using lactate dehydrogenase and Enzyme-linked ImmunoSpot assays, respectively. The results from the present study revealed that the expression of IFN-γ, interleukin (IL)-2, tumor necrosis factor-α and IL-12 of PBMCs from patients with LC and healthy donors were significantly increased following treatment with rMBP-NAP (P<0.05). Additionally, rMBP-NAP significantly upregulated the number of IFN-γ-secreting cells in PBMCs and prominently increased the cytotoxic activity of PBMCs (P<0.05). Furthermore, the expression of TLR2 was significantly enhanced following rMBP-NAP stimulation (P<0.05), which indicated that rMBP-NAP may serve an antitumor role via TLR2 signaling pathways. Overall, these results demonstrated that rMBP-NAP possesses the potential to be a novel immunomodulatory candidate drug and requires further evaluation in clinical trials.

Recombinant protein rMBP-NAP restricts tumor progression by triggering antitumor immunity in mouse metastatic lung cancer

Recombinant Helicobacter pylori neutrophil-activating protein fused with maltose-binding protein (rMBP-NAP), a potential TLR2 ligand, was reported to possess immunomodulatory effects on in situ tumors in our previous study. In the present work, we attempt to elucidate the effect of rMBP-NAP at the local immune modulation in B16-F10-induced metastatic lung cancer. Our results demonstrated that growth of B16-F10 melanoma metastases in the lung was significantly arrested after rMBP-NAP treatment, along with marked reduction in metastatic lung nodules and significant increase in survival. The treatment induced both local and systemic immune responses, which were associated with higher influx of CD4+/CD8+ T cells and drove toward Th1-like and cytotoxic immune environment. Moreover, rMBP-NAP also showed significant anti-angiogenic activity by reducing vascularization in lung tumor sections. rMBP-NAP could induce antitumor immunity through activating Th1 cells and producing pro-inflammatory cytokines, which are responsible for the effective cytotoxic immune response against cancer progression. Our findings indicate that rMBP-NAP might be a novel antitumor therapeutic strategy.

Protein 4.1R attenuates autoreactivity in experimental autoimmune encephalomyelitis by suppressing CD4(+) T cell activation.

Immune synapse components contribute to multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE) pathogenesis as they play important role in autoreactive T cell activation. Protein 4.1R, a red cell membrane cytoskeletal protein, recently was identified as an important component of immunological synapse (IS) and acted as the negative regulator of CD4(+) T cell activation. However, the pathological role of 4.1R in the MS/EAE pathogenesis is still not elucidated. In this study, we investigated the potential role of protein 4.1R in pathologic processes of EAE by using 4.1R knockout mouse model. Our results suggest that 4.1R can prevent pathogenic autoimmunity in MS/EAE progression by suppressing the CD4(+) T cell activation.