Chrysanthemum sporopollenin: A novel vaccine delivery system for nasal mucosal immunity.

Objective: Mucosal immunization was an effective defender against pathogens. Nasal vaccines could activate both systemic and mucosal immunity to trigger protective immune responses. However, due to the weak immunogenicity of nasal vaccines and the lack of appropriate antigen carriers, very few nasal vaccines have been clinically approved for human use, which was a major barrier to the development of nasal vaccines. Plant-derived adjuvants are promising candidates for vaccine delivery systems due to their relatively safe immunogenic properties. In particular, the distinctive structure of pollen was beneficial to the stability and retention of antigen in the nasal mucosa. Methods: Herein, a novel wild-type chrysanthemum sporopollenin vaccine delivery system loaded with a w/o/w emulsion containing squalane and protein antigen was fabricated. The unique internal cavities and the rigid external walls within the sporopollenin skeleton construction could preserve and stabilize the inner proteins. The external morphological characteristics were suitable for nasal mucosal administration with high adhesion and retention. Results: Secretory IgA antibodies in the nasal mucosa can be induced by the w/o/w emulsion with the chrysanthemum sporopollenin vaccine delivery system. Moreover, the nasal adjuvants produce a stronger humoral response (IgA and IgG) compared to squalene emulsion adjuvant. Mucosal adjuvant benefited primarily from prolongation of antigens in the nasal cavity, improvement of antigen penetration in the submucosa and promotion of CD8+ T cells in spleen. Disccusion: Based on effective delivering both the adjuvant and the antigen, the increase of protein antigen stability and the realization of mucosal retention, the chrysanthemum sporopollenin vaccine delivery system has the potential to be a promising adjuvant platform. This work provide a novel idea for the fabrication of protein-mucosal delivery vaccine.

EphA3 contributes to tumor growth and angiogenesis in human gastric cancer cells.

Eph receptor tyrosine kinases and their ephrin ligands, mediate an important cell communication system both in normal and oncogenic development, and play central roles in a series of processes including angiogenesis, stem cell maintenance and cancer metastasis. Eph receptor A3 (EphA3), commonly overexpressed in a broad range of cancers, including gastric cancer (GC), is related to tumor progression. Our previous study revealed that EphA3 may play important roles in tumorigenesis and angiogenesis in GC. However, its exact role and the mechanisms underlying its function in GC remain unclear. In the present study, lentivirus­mediated RNA interference was employed to knock down the expression of EphA3 in GC HGC­27 cells. Functional analyses indicated that depletion of EphA3 expression inhibited the cell growth and tumorigenicity of HGC­27 cells in vitro and in vivo. Furthermore, knockdown of the expression of EphA3 in HGC­27 cells inhibited tube formation and migration of HUVEC endothelial cells. Tumor angiogenesis in vivo was also inhibited upon EphA3 knockdown in HGC­27 cells, with reduced microvessel density (MVD) in xenograft models. We further revealed that EphA3 depletion inhibited tumor angiogenesis and migration through the signal transducer and activator of transcription 3/vascular endothelial growth factor (STAT3/VEGF) signaling pathway. These results indicated that EphA3 may be an effective prognostic indicator and a potential target for GC therapy.

Analysis of differentially expressed genes in LNCaP prostate cancer progression model.

The LNCaP/C4-2 human prostate cancer progression model was established to mimic phenotypic and genotypic changes during prostate cancer development from androgen dependence to androgen independence, from nonmetastasis to metastasis. In this study, cDNA microarrays were performed using a microarray chip from Affymetrix to characterize and compare gene expression profiles in LNCaP and C4-2, which may provide novel insight into the molecular mechanism mediating prostate cancer progression. Three hundred eighteen genes consistently exhibited differential expression in LNCaP and C4-2 in 2-time microarray data. Based on their function, the differentially expressed genes can be grouped into several subcategories, including growth factors and signal transducers, oncogenes and tumor suppressors, tumor-specific antigens, transcriptional factors, transporters, and factors involved in invasion, metastasis, and metabolism. Some genes are novel and unexplored in prostate cancer progression and are of potential interest for follow-up investigation. Reverse transcription-polymerase chain reaction (RT-PCR) and real-time RT-PCR were performed to corroborate the microarray results, and 76 differentially expressed genes were validated out of 104 candidates. Expression pattern analyses were performed in these 76 differentially expressed genes, and a series of genes was found to be positively or negatively correlated to prostate cancer progression in the LNCaP prostate cancer progression model and to possess predominant prostate cell specificity. ELF5/ESE-2b and long-chain acyl coenzyme A dehydrogenase (ACADL) expressions were found to be positively associated with malignant progression in LNCaP, C4-2, and C4-2B, and predominantly expressed in prostate cancer cells. Functional evaluation revealed that ELF5/ESE-2b and ACADL expressions contributed to the malignant phenotypes of prostate cancer cells. Accordingly, our microarray data may provide clues for finding novel genes involved in prostate cancer progression to androgen independent and metastasis, and shed light on finding new targets for diagnosis and therapy of prostate cancer.

[PC-1 enhances c-myc gene expression in prostate cancer cells].

PC-1(Prostate and colon gene 1) gene belongs to TPD52 (Tumor Protein D52) gene family. The expression of PC-1 is found to promote androgen-independent progression. This study was conducted to assess the mechnism of promotion of androgen-independent progression in PC-1 gene. The c-myc gene expression was tested by RT-PCR and Western blotting analyses in the LNCaP-pc-1 and LNCaP-zero cell line. After separation of cytoplasm and nulear proteins of the LNCaP-pc-1 and LNCaP-zero cell line, the beta-catenin protein was detected by Western blotting. C4-2 cell line was used to examine the effects of 10058-F4 on the PC-1 gene expression. The results of RT-PCR and Western blotting indicated that PC-1 enhanced c-myc gene expression in prostate cancer cells, PC-1 was also found to enhance beta-catenin expression in nuclear. Furthermore, a small-molecule c-Myc inhibitor, 10058-F4 represses PC-1 gene expression in C4-2 cell line. Our findings suggest that PC-1 enhances c-myc gene expression in prostate cancer cells through the Wnt/beta-catenin pathway. Meanwhile, c-myc plays a feed-forward role in enhancing PC-1 driven c-myc gene expression, and promotes prostate an-drogen-independent progression.

[Identification and expression of two new secretory proteins associated with prostate cancer].

Our research intends to obtain extra-cellular proteinogram of cell lines representing different advancement stages of prostate cancer and to test whether screened differential expression proteins can be secreted and used as serum biomarkers for prostate cancer. By examining differential expression spots in two extra-cellular protein 2D-PAGE gels and mass spectrum, candidate molecules were obtained. The expressions of these candidate molecules in eight cell lines and response to androgen stimulus in LNCaP were analyzed by RT-PCR. By constructing eukaryotic expression vectors and western-blotting with anti tags antibodies, the candidate molecules were tested to understand whether they can be expressed in transfected 293T cell culture fluid. Two overexpressed molecules-triosephosphate isomerase 1 (TPI1) and syndecan bind-ing protein, syntenin (ST1)-in extra-cellular proteinogram of C4-2 were screened out; both of them are secretary proteins. On transcriptional level, both proteins were up-regulated with the malignancy of prostate cancer cell lines and ST1 was dose-dependently inhibited by androgen. Considering cellular level results, both TPI1 and ST1 have their potential as serum biomarkers for indicating the developmental stage of prostate cancer.

The radiation response of androgen-refractory prostate cancer cell line C4-2 derived from androgen-sensitive cell line LNCaP.

Radiation therapy is a relatively effective therapeutic method for localized prostate cancer (PCa) patients. However, radioresistance occurs in nearly 30% of patients treated with potentially curative doses. Therapeutic synergy between radiotherapy and androgen ablation treatment provides a promising strategy for improving the clinical outcome. Accordingly, the androgen deprivation-induced signaling pathway may also mediate radiosensitivity in PCa cells. The C4-2 cell line was derived from the androgen-sensitive LNCaP parent line under androgen-depleted condition and had acquired androgen-refractory characteristics. In our study, the response to radiation was evaluated in both LNCaP and C4-2. Results showed that C4-2 cells were more likely to survive from irradiation and appeared more aggressive in their resistance to radiation treatment compared with LNCaP, as measured by clonogenic assays and cell viability and cell cycle analyses. Gene expression analyses revealed that a set of genes involved in cell cycle arrest and DNA repair were differentially regulated in LNCaP and C4-2 in response to radiation, which was also consistent with the radiation-resistant property observed in C4-2 cells. These results strongly suggested that the radiation-resistant property may develop with progression of PCa to androgen-independent status. Not only can the LNCaP and C4-2 PCa progression model be applied for investigating androgen-refractory progression, but it can also be used to explore the development of radiation resistance in PCa.

Ubiquitous mitochondrial creatine kinase is overexpressed in the conditioned medium and the extract of LNCaP lineaged androgen independent cell lines and facilitates prostate cancer progression.

BACKGROUND: Androgen independent prostate cancer (AIPC) is not responsive to androgen ablation therapy. The biomarkers of AIPC are lack. Numerous proteomics studies have focused on finding new markers of AIPC and exploring their possible functions, but little is known about the difference between conditioned medium (CM) from AIPC and androgen dependent prostate cancer (ADPC) cells. METHODS: We performed a proteome analysis of CM from LNCaP, C4-2, and C4-2B cells by a two dimensional electrophoresis based technology. Western blots and immunohistochemical studies were employed to explore the expression pattern of the identified protein in prostate cancer cell lines and clinical specimens, respectively. Then we examined the possible roles and mechanisms of the ubiquitous mitochondrial creatine kinase (uMtCK) in vitro. RESULTS: Besides prostate specific antigen (PSA) and insulin-like growth factor binding protein-2 (IGFBP2), uMtCK was identified in the CM of AIPC cells. uMtCK was up-regulated in AIPC cells and in human prostate cancer tissues at WHO grade III. Stably transfected exogenous uMtCK showed a growth promoting effect rather than mock vector in LNCaP cells, with or without bicalutamide in culture medium. Further assays showed that higher degrees of ROS generation and Akt signaling pathway activation in LNCaP-uMtCK than in LNCaP-neo cells. CONCLUSIONS: We showed that uMtCK could be easily detected in CM of LNCaP lineaged AIPC cells. Exogenous uMtCK in LNCaP cells surprisingly contributed to overproduction of ROS, activation of Akt signaling pathway and more aggressive phenotypes including androgen independence development.

[FTIR study of adsorption of PCP on hematite surface].

The adsorption of pentachlorophenol on hematite was studied through adsorption experiments and FTIR analysis. The pH adsorption isotherms of pentachlorophenol onto hematite were obtained by the static state experiments. The largest adsorption quantity occurred at about pH 6. The adsorption quantity at pH 8.5 of the isoelectric point of hematite was about 31% of the largest adsorption quantity. Fourier transform infrared (FTIR) spectroscopy was used to analyse the change of hematite before and after PCP adsorption, and the species of PCP on hematite. It was discovered that: (1) the typical peak at 565 cm(-1) of the Fe-O bond in alpha-Fe2O3 did not change before and after adsorption, and the adsorption occurred on the surface of hematite. (2) At pH 6.0, the stretching vibration peak at 3 438 cm(-1) due to the hydrogen bond formed between O-H on the surface of alpha-Fe2O3 and water molecules shifted to 3 417 cm(-1). The bending vibration peak of H-O-H+ on the surface at 1 643 cm(-1) was weakened because of complex reaction. The peak owing to Fe-OH bond was displaced from 1 050-1 100 cm(-1) to 950 cm(-1) with increased intensity. The C-O bond stretching vibration peak of PCP was displaced from 1 215 to 1 122 cm(-1). The main interaction between PCP and hematite was static electric interaction. (3) At pH 8.5, the stretching vibration peak of the hydrogen bond formed between O-H on the surface of alpha-Fe2O3 and water molecules was displaced from 3 438 to 3 428 cm(-1). The bending vibration peak at 1 643 cm(-1) was obviously weakened because of the hydrogen bonding. The H-O-H+ bending vibration peak at 1 050-1 100 cm(-1) was displaced to 947 cm(-1) with obviously increased intensity, indicating that the interaction was mainly through hydrogen bond.

[Gene fusion of egfp & kan and recombinant plasmid construction by red mediated in vivo homologous recombination].

Recombineering, a new genetic engineering technology based on high efficiency in vivo homologous recombination, can be used in target DNA knock-in, knock-out and gene cloning. In the process of gene subcloning mediated by Recombineering technique, high-quality target DNA fragments were difficult to obtain using in vitro overlapping PCR,therefore the efficiency of in vivo homologous recombination was severely interrupted. To solve this problem, some technology improvements have been established based on the principle of Red recombinases. The PCR DNA fragments of egfp and kan genes with complementary sequences on the end of each fragment were co-introduced into a pcDNA3.1 vector and Red recombinases containing E. coli DY331 host cells by electroporation. A recombinant plasmid pcDNA3.1-egfp-kan was screened directly by antibiotic marker. The positive rates can reach to 45%. The EGFP gene expression of pcDNA3.1-egfp-kan can be observed by transient transfection of 293 eukaryotic cells.

[pBR322-Red mediated gene knockin, sites and expression in E. coli chromosome].

Genes lacZ, lacY and lacA in the lac opron of E. coli chromosome were respectively substituted with gene luc by using plasmid pBR322-Red, selection-counterselection system kan/sacB and various strategies of Red homologous recombination including Red mediated linearized double-stranded DNA homologous recombination and Red mediated recombineering with overlapping single stranded DNA oligonucleotides. Then, a series of new strains, CWL2, CWL4 and CWL6, were constructed and we found that they can express protein Luc efficiently. To further study the expression of exogenous genes at the site of lacZ, we have constructed a strain named CWD1 by knockin the cholera toxin B subunit(ctxb) gene at the lacZ site, then we found that CWD1 can express protein CTB efficiently and CTB was secreted out of the cell. So we assured that the sites of structure genes in the lac operon of Escherichia coli chromosome were suitable for expressing foreign genes.

[Construction of recombinant plasmid using Neo/E Technology].

A new neo/E counterselection technique was set up using Red recombination, which could be used in constructing recombinant plasmid. Firstly, linear targeting cassettes were amplified by PCR; secondly, two steps of homology recombination occurred in vivo: (1) The neo/E counterselection targeting cassette, consisting of a unique endonuclease recognition site and an antibiotic resistance gene, was introduced into the targeted region. (2) The neo/E cassette was replaced in the second round of recombination by another linear targeting cassettes DNA fragment carrying the targeted gene. For selecting a correct recombinant plasmid from the mixture of nonrecombinant and recombinant clones, the unique endonuclease recognition site in the nonrecombinant clones was cut by endonuclease and then transformed into the E. coli competent cells, up to 20% correct recombinants were yielded. A recombinant plasmid of pGL3-Basic PC1900T was successfully constructed in this way. Application of this technique offers a new and highly efficient way for recombinant plasmids construction.

[Development of a new recombineering system by gap repair].

Using lambda phage Red recombinase mediated in vivo homologous recombination system, a 6.7 kb lambda PL operon sequence including the Red encoding genes was subcloned into pBR322 by gap repair technique, and generated a pBR322-Red recombinant plasmid that can provide the Red recombination function and can be transferred into many kinds of bacteria. To confirm the recombination functions of pBR322-Red, a single-stranded 70-bases oligo was introduced into W3110 by electroporation to create a single base T-->G mutation in galK gene on the bacterial chromosome. The result demonstrated that a new lambda Red-mediated recombineering system based on pBR322-Red was successfully established.

[Gene knockout and knockin on the Escherichia coli lac operon loci using pBR322-red system].

pBR322-Red is a newly constructed recombineering plasmid, which contains a part of the pBR322 vector, a series of regulatory elements of lambda-prophage and Red recombination genes. In the beginning, we studied the best working conditions of pBR322-Red, and then modified lac operon in E. coli W3110 chromosome using the plasmid as follow: Firstly, we knockout the lacI gene using Red-mediated recombineering with overlapping single stranded DNA oligonucleotides. Secondly, we substituded the lacA and lacY genes with lacZ, a report gene, by Red-mediated linearized double strands DNA homologous recombination. Finally, we detected the expression of lacZ on these loci for the first time. The results suggested that pBR322-Red system is suitable for modifying W3110 chromosome with various recombination strategies.