PD-L1 siRNA hitched polyethyleneimine-elastase constituting nanovesicle induces tumor immunogenicity and PD-L1 silencing for synergistic antitumor immunotherapy.

BACKGROUND: PD-1/PD-L1 blockade has become a powerful method to treat malignant tumors. However, a large proportion of patients still do not benefit from this treatment, due to low tumor immunogenicity and low tumor penetration of the agents. Recently, neutrophil elastase has been shown to induce robust tumor immunogenicity, while the insufficient enzyme activity at the tumor site restricted its anti-tumor application. Here, we designed polyethyleneimine-modified neutrophil elastase (PEI-elastase) loaded with PD-L1small interfering RNA (PD-L1 siRNA) for improving enzymatic activity and delivering siRNA to tumor, which was expected to solve the above-mentioned problems. RESULTS: We first demonstrated that PEI-elastase possessed high enzymatic activity, which was also identified as an excellent gene-delivery material. Then, we synthesized anti-tumor lipopolymer (P-E/S Lip) by encapsulating PEI-elastase and PD-L1siRNA with pH-responsive anionic liposomes. The P-E/S Lip could be rapidly cleaved in tumor acidic environment, leading to exposure of the PEI-elastase/PD-L1 siRNA. Consequently, PEI-elastase induced powerful tumor immunogenicity upon direct tumor killing with minimal toxicity to normal cells. In parallel, PEI-elastase delivered PD-L1siRNA into the tumor and reduced PD-L1 expression. Orthotopic tumor administration of P-E/S Lip not only attenuated primary tumor growth, but also produced systemic anti-tumor immune response to inhibit growth of distant tumors and metastasis. Moreover, intravenous administration of P-E/S Lip into mice bearing subcutaneous tumors leaded to an effective inhibition of established B16-F10 tumor and 4T1 tumor, with histological analyses indicating an absence of detectable toxicity. CONCLUSIONS: In our study, a protease-based nanoplatform was used to cooperatively provoke robust tumor immunogenicity and down-regulate PD-L1 expression, which exhibited great potential as a combination therapy for precisely treating solid tumors.

Potential role of Wnt/ß-catenin signaling in blastic transformation of chronic myeloid leukemia: cross talk between ß-catenin and BCR-ABL.

Chronic myeloid leukemia (CML) results from malignant transformation of hematopoietic stem cells induced by the BCR-ABL oncogene. Transformation from chronic to blastic phase is the lethal step in CML. Leukemic stem cells (LSCs) are the basic reason for blastic transformation. It has been shown that Wnt/ß-catenin signaling contributes to the self-renewal capacity and proliferation of LSCs in CML. However, the role of Wnt/ß-catenin signaling in blastic transformation of CML is still obscure. Here, we explored the relationship between BCR-ABL and ß-catenin signaling in vitro and in vivo. We found that BCR-ABL stimulated ß-catenin via activation of PI3K/AKT signaling in blastic phase CML cells. Inhibition of the kinase activity of BCR-ABL, PI3K, or AKT decreased the level of ß-catenin in both K562 cells and a CML mouse model and suppressed the transcription of downstream target genes (c-myc and cyclin D1). In addition, inhibition of the BCR-ABL/PI3K/AKT pathway delayed the disease progression in the CML mouse model. To further explore the role of ß-catenin in the self-renewal and survival of CML LSCs, we established a secondary transplantation CML mouse model. Our data revealed that inhibition of the BCR-ABL/PI3K/AKT pathway reduced the tumor-initiating ability of K562 cells, decreased leukemia cell infiltration into peripheral blood and bone marrow, and prolonged the survival of mice. In conclusion, our data indicate a close relationship between ß-catenin and BCR-ABL/PI3K/AKT in blastic phase CML. ß-Catenin inhibition may be of therapeutic value by targeting LSCs in combination with a tyrosine kinase inhibitor, which may delay blastic transformation of CML.

[Effect of indomethacin on BCR/ABL-Wnt/ß-catenin pathway in K562 cells].

OBJECTIVE: To investigate the effect of indomethacin on the proliferation and Wnt/ß-catenin pathway in K562 cells. METHODS: The cell growth of K562 cells treated with different concentrations of indomethacin was assessed with MTT assay, and the colony-forming ability of the cells was evaluated by colony-forming assay. The mRNA expressions of BCR/ABL and ß-catenin were detected by RT-PCR, and the protein expressions of pBCR/ABL, total BCR/ABL, ß-catenin, pGSK-3ß and c-myc were analyzed by Western blotting. RESULTS: Indomethacin significantly suppressed the growth and colony-forming ability of K562 cells in a dose-dependent manner. Indomethacin treatment dose-dependently decreased the protein level of pBCR/ABL and total BCR/ABL without affecting bcr-abl mRNA expressions. Compared with the control groups, indomethacin-treated cells showed obviously decreased mRNA and protein expressions of ß-catenin and decreased protein expressions of pGSK-3ß and c-myc. CONCLUSION: Indomethacin inhibits the proliferation of K562 cells by suppressing the activity of bcr-abl-Wnt/ß-catenin pathway.

[Indomethacin Significantly Enhances Inhibitory Effect of Imatinib on Proliferation of KCL22 and K562/G01 Cells].

OBJECTIVE: To investigate the inhibitory effect of indomethacin combined with imatinib on proliferation of KCL22 and K562/G01 cells, and to elucidate the molecular mechanism of antiproliferative effect by Wnt/ß-Catenin signaling way. METHODS: Indomethacin was used in KCL22 and K562/G01 cells. The cell growth was detected by MTT assay to explore the optimal concentration and time. The effect of drugs on proliferation capacity was assessed by MTT assay and colony-forming assay. Flow cytometry was used to identify the cell cycle and apoptosis changes. The protein expression of pß-catenin (S33/37/T41), pGSK-3ß (Ser9) and C-MYC were analyzed by Western blot. RESULTS: The optimal concentration and time of indomethacin on KCL22 and K562/G01 were 80 µmol/L for 48 h. The inhibitory effect of 80 µmol/L indomethacin combined 2 µmol/L imatinib on cell proliferation was significantly better than a single drug treatment. Flow cytometry results showed that cell cycle was arrested in the G0/G1 phase in both combined treatment groups. The number of apoptosis cells in combined treatment groups was significantly higher than that in single drug treatment groups. Compared with the control group or single drug treatment groups, the protein level of pß-catenin, ß-catenin, pGSK-3ß (Ser9) and C-MYC decreased significantly. CONCLUSION: Indomethacin significantly enhances inhibitory effect of imatinib on proliferation of KCL22 and K562/G01 cells and regulate cell proliferation through Wnt/ß-Catenin signaling way.

Graphene sheets, polyaniline and AuNPs based DNA sensor for electrochemical determination of BCR/ABL fusion gene with functional hairpin probe.

A sensitive and selective electrochemical DNA sensor was developed for the detection of BCR/ABL fusion gene in chronic myelogenous leukemia (CML). Firstly, graphene sheets (GS) suspension was prepared with the aid of chitosan (CS) solution and then fabricated onto the glassy carbon electrode (GCE), followed by the electro-polymerization of aniline to form the PANI layer, then, Au nanoparticles (AuNPs) were electro-deposited onto the modified GCE to immobilize the capture probes. The capture probe employed a hairpin structure and dually labeled with a 5'-SH and a 3'-biotin. After hybridization with the target DNA, hairpin structure was compelled to open and 3'-biotin was forced to stay away from the electrode surface. As a result, streptavidin-alkaline phosphatase (SA-AP) was covalently binded to the capture probe via biotin-avidin system. Reduction currents were then generated after catalyzing the hydrolysis of the electroinactive 1-naphthyl phosphate (1-NP) to 1-naphthol and monitored by differential pulse voltammetry (DPV). Under optimum conditions, the amperometric signals increased linearly with the target DNA concentrations (10 pM to 1000 pM), and the DNA sensor exhibited a detection limit as low as 2.11 pM (S/N=3) with an excellent differentiation ability, and the proposed method showed acceptable stability and reproducibility. It has been applied for assay of BCR/ABL fusion gene from real samples with satisfactory results.