Construction of a novel fusion protein harboring mouse inter- feron γ and epidermal growth factor receptor binding domain and enhancement of its antitumor activity

A novel fusion protein harboring mouse interferon γ and epidermal growth factor receptor binding domain was constructed with the method of genetic and protein engineering. The fusion protein kept complete antiviral activity with the titer of 108 IU per liter of culture. The EGF-RBD of the fusion protein exhibited competitive binding activity against 125I-mEGF for mEGF receptors on A431 cells. The fusion protein was shown to be more potent in in-hibiting the growth of cultured mouse breast carcinoma cells than interferon γ. Experimental data on mouse B16 malig-nant melanoma model indicated that the tumor weight of fusion protein-treated group was statistically significantly smaller than that of interferon γ-treated group. The work here provides a necessarily reliable clue for the upcoming clinical employment of a novel class of targeting interferons.     

The antibody preparation and expression of human Pescadillo

To explore the biological roles of human Pescadillo and investigate its potential effect on tumorigene- sis, the cDNA of Pescadillo was fused with that of GST. After purification and elution, the purified GST-Pescadillo fusion protein was obtained, and the antibody against the fusion protein was generated. Endogenous Pescadillo protein was observed to be remarkably induced by estrogen. It was mainly distributed in the tissues such as breast, ovary and intestine, all of which contain proliferating cells, and was also detected in many cell lines of human cancer: renal carcinoma, hepatoma, ovarian cancer, colon carcinoma, and breast cancer. The expression level of Pescadillo was increased significantly in breast cancer tissues compared with their paired margin tissues. Taken together, these data suggest that Pescadillo may play important roles in the initiation and development of cancer and may be a po- tential target in cancer diagnosis and therapy.     

Preparation and evaluation of a new releasable PEGylated tumor necrosis factor-α(TNF-α) conjugate for therapeutic application

To design a releasable PEGylated TNF-α(rPEG-TNF-α ), a cathepsin B-sensitive dipeptide (Val-Cit moiety) was inserted into conventional PEG-modified TNF- (PEG-TNF- ), facilitating its clinical use for anti-tumor therapy. Comparative pharmaco- kinetic and pharmacodynamic studies showed that the half-lives of both PEGylated forms of TNF-α were ~60-fold greater than that of unmodified TNF-α . In addition, the in vitro bioactivity of rPEG-TNF-α was greater than that of PEG-TNF-α with the same degree of PEG modification. Release of TNF-α from rPEG-TNF-α in vitro was dependent on the presence of cathepsin B and was inhibited by a cathepsin B inhibitor. Despite the potent cytotoxicity of unmodified TNF-α against normal cells, its PEGylated forms at higher TNF-α concentrations showed low cytotoxic activity against these cells. In contrast, both forms of PEGylated TNF-α showed potent cytotoxic activity against the B16 and L929 cell lines, with rPEG-TNF-α being 5- and 9- fold more potent, respectively, than PEG-TNF-α . Moreover, rPEG-TNF-α was a more potent in vivo antitumor agent than PEG-TNF-α .     

Construction,expression and characterization of the engineered antibody against tumor surface antigen,p185^c—erbB—2

The c-erbB-2 proto-oncogene encodes a 185kD protein p185,which belongs to epidermal growth factor receptor family.Amplification of this gene has been shown to correlate with poor clinical prognosis for certain cancer patients.The monoclonal antibody A21 which directed against p185 specifically inhibits proliferation of tumor cells overexpressing p185,hence allows it to be a candidate for targeted therapy.In order to overcome several drawbacks of murine MAb,we cloned its VH and VL genes and constructed the single-chain FV(scFv)through a peptide linker.The recombinant scFv A21 was expressed in Escherichia coli and purified by the affinity column.Subsequently it was characterized by ELISA,Western blot,cell immunohistochemistry and FACS.All these assays showed the binding activity to extracellular domain(ECD)of p185.Based on those properties of scFvA21,we further constructed the scFv-Fc fusion molecule with a homodimer form and the recombinant product was expressed in mammalian cells.In a series of subsequent analysis this fusion protein showed identical antigen binding site and activity with the parent antibody.These anti-p185 engineered antibodies have promised to be further modified as a tumor targeting drugs,with a view of application in the diagnosis and treatment of human breast cancer.     

Casticin suppresses self-renewal and invasion of lung cancer stem-like cells from A549 cells through down-regulation of pAkt

A subpopulation of cancer stem cells is recognized as the cause of tumorigenesis and spreading. To investigate the effects of casticin （5,3＇-dihydroxy-3,6,7,4＇-tetramethoxyflavone）, derived from Fructus Viticis Simplicifoliae, on lung cancer stem cells, we isolated and identified a subpopulation of lung cancer stem-like cells （LCSLCs） from non-small-cell lung carcinoma A549 cells with the features including self- renewal capacity and high invasiveness in vitro, elevated tumorigenic activity in vivo, and high expression of stemness markers CD133, CD44, and aldehyde dehydrogenase 1 （ALDH1）, using serum-free suspension sphere-forming culture method. We then found that casticin could suppress the proliferation of LCSLCs in a concentration-dependent manner with an IC50 value of 0.4 μmol/L, being much stronger than that in parental A549 cells. In addition, casti- cin could suppress the self-renewal and invasion of LCSLCs concomitant with decreased CD133, CD44, and ALDH1 protein expression and reduced MMP-9 activity. Further experiments showed that casticin suppressed self-renewal and invasion at least partly through down-regulation of Akt phosphorylation. In conclusion, casticin suppressed the characteristics of LCSLCs, suggesting that casticin may be a candidate compound for curing lung cancer via elim- inating cancer stem cells.     

Vegetative Storage Protein with Trypsin Inhibitor Activity Occurs in Sapindus mukorassi,a Sapindaceae Deciduous Tree

A vegetative storage protein （VSP） with trypsin inhibitor activity in a deciduous tree, Sapindus mukorassi, was characterized by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western-blot, immuno-histochemical localization, light- and electro-microscopy, together with analysis of proteinase inhibitor activity of the purified VSP in vitro. There were two proteins with molecular masses of about 23 and 27 kDa in a relatively high content in the bark tissues of terminal branches of S. mukorassi in leafless periods. The proteins decreased markedly during young shoot development, indicating their role in seasonal nitrogen storage. Immuno-histochemical localization with the polyclonal antibodies raised against the 23 kDa protein demonstrated that the 23 kDa protein was the major component of protein inclusions in protein-storing cells. The protein inclusions were identified by protein-specific staining and should correspond to the electron-dense materials in different forms in the vacuoles of phloem parenchyma cells and phloem ray parenchyma cells under an electron microscope. So, the 23 kDa protein was a typical VSP in S. mukorassi. The 23 and 27 kDa proteins shared no immuno-relatedness, whereas the 23 kDa protein was immuno-related with the 22 kDa VSP in lychee and possessed trypsin inhibitor activity. The 23 kDa protein may confer dual functions： nitrogen storage and defense.     

Novel benzenediamine derivative FC99 ameliorates zymosan-induced arthritis by inhibiting RORγt expression and Thl7 cell differentiation

Increased IL-17-producing helper T （Thl7） cells have been observed in patients with rheumatoid arthritis （RA）. The retinoic-acid-related orphan nuclear receptor （RORγt） is the master regulator of Thl7 cells. Our previous research showed that FC99 possesses anti-inflammation activity. However, to date the effects of FC99 on RORγt expression in Thl7 cell differentiation have not been investigated yet. In the present study, we found that FC99 significantly attenu- ated arthritis-like symptoms, i.e., suppressing the develop- ment of paw edema in zymosan-induced arthritis （ZIA） mice. H＆E staining showed that the infdtration of inflamma- tory cells in ankle synovial tissues was significantly suppressed. FC99 also reduced the mRNA levels of pro-in- flammatory cytoklnes in ankle synovial tissues as shown by Q-PCR analysis. The protein levels of the pro-inflammatory cytoklnes in sera were also suppressed after FC99 treatment. Moreover, FC99 decreased the RORγt mRNA level in spleen tissues. Thl7 cell percentage was significantly decreased in spleens and draining lymph nodes （dLNs）. The mRNA and protein levels of IL-17A and IL-23 were reduced after FC99 treatment in ZIA mice. Furthermore, in vitro experiments showed that FC99 inhibited the expression of IL-6 in LPS- induced RAW264.7 cells and BMDCs. Moreover, FC99 sig- nificantly inhibited the RORγt expression in PMA-induced CD4＋ T cells and LPS-induced RAW264.7 cells. These data indicate that FC99 improves arthritis-like pathological symp- toms in vivo and in vitro, which might be related to the inhib- ition of RORγt expression in Thl7 cells. Our findings suggest that FC99 may be a potential therapeutic candidate for the treatment of RA and other inflammatory disorders.     

Glycogen synthase kinase-3β positively regulates the proliferation of human ovarian cancer cells

Although glycogen synthase kinase-3 （GSK-3） might act as a tumor suppressor since its inhibition is expected to mimic the activation of Wnt-signaling pathway, GSK-3β may contribute to NF-κB activation in cancer cells leading to increased cancer cell proliferation and survival. Here we report that GSK-3β activity was involved in the proliferation of human ovarian cancer cell both in vitro and in vivo. Inhibition of GSK-3 activity by pharmacological inhibitors suppressed proliferation of the ovarian cancer cells. Overexpressing constitutively active form of GSK-3β induced entry into the S phase, increased cyclin D1 expression and facilitated the proliferation of ovarian cancer cells. Furthermore, GSK-3 inhibition prevented the formation of the tumor in nude mice generated by the inoculation of human ovarian cancer cells. Our findings thus suggest that GSK-3β activity is important for the proliferation of ovarian cancer cells, implicating this kinase as a potential therapeutic target in ovarian cancer.     

Purification and characterization of a mannose-binding lectin from the rhizomes of Aspidistra elatior Blume with antiproliferative activity

A lectin with a novel N-terminal amino acid sequence was purified from the rhizomes of Aspidistra elatior Blume by ammonium sulphate precipitation, ion exchange chromatography on diethylaminoethyl-Sepharose and carboxymethyl-Sepharose and gel filtration chromatography on Sephacryl S-100. The A. elatior Blume lectin （AEL） is a heterotetramer with a molecular mass of 56 kDa and composed of two homodimers consisting of two different polypeptides of 13.5 kDa and 14.5 kDa held together by noncovalent interactions. Hapten inhibition assay indicated that hemagglutinating activity of AEL towards rabbit erythrocytes could be inhibited by D-mannose, mannan, thyroglobulin and ovomucoid. The lectin was stable up to 70 ℃ , and showed maximum activity in a narrow pH range of 7.0-8.0. Chemical modification and spectrum analysis indicated that tryptophan, arginine, cysteine and carboxyl group residues were essential for its hemagglutinating activity. However, they might not be present in the active center, except some carboxyl group residues. AEL also showed significant in vitro antiproliferative activity towards Bre-04 （66%）, Lu-04 （60%） and HepG2 （56%） of human cancer cell lines.     

Antioxidant activity of mangostin in cell-free system and its effect on K562 leukemia cell line in photodynamic therapy

Mangostin （MAG）, a kind of xanthone widely used in diet and medicine, has antioxidant, anti-inflammatory, antimicrobial, and anticancer activities. On account of its antioxidant activity, MAG might protect cancer cells from free radical damage in photodynamic therapy （PDT） during which reactive oxygen species production was stimulated leading to irreversible tumor cell injury. In this study, the antioxidant activity of MAG was investigated and the influence of MAG on K562 cells in 5-aminolevulinic acid （ALA）-based PDT is demon- strated. The results showed that MAG could scavenge hydroxyl radical, superoxide anion, and hydrogen per- oxide and inhibit the formation of malondialdehyde （MDA）, but increase the amounts of singlet oxygen in cell-free systems. MAG inhibits cell proliferation and enhances cell apoptosis, lipid peroxidation, and DNA damage in ALA-PDT on K562 cells. NAN3, a singlet oxygen quencher, suppresses the MAG-induced cell apoptosis, lipid peroxidation, and DNA damage. In con- clusion, MAG enhances the PDT-induced cytotoxicity in K562 cells and singlet oxygen was involved in this process. These results implied that the effect of antioxidants on PDT might be determined by its sensitization ability to singlet oxygen.     

Fucoxanthin induces growth arrest and apoptosis in human bladder cancer T24 cells by up-regulation of p21 and down-regulation of mortalin

Fucoxanthin, a natural carotenoid, has been reported to have anti-cancer activity in human colon cancer cells, human prostate cancer cells, human leukemia cells, and human epithelial cervical cancer cells. This study was undertaken to evaluate the molecular mechanisms of fuco- xanthin against human bladder cancer T24 cell line. MTT analysis results showed that 5 and 10 ixM fucoxanthin inhibited the proliferation of T24 cells in a dose- and time- dependent manner accompanied by the growth arrest at Go/G1 phase of cell cycle, which is mediated by the up-regu- lation of p21, a cyclin-dependent kinase （CDK）-inhibitory protein and the down-regulation of CDK-2, CDK-4, cyclin D1, and cyclin E. In addition, 20 and 40 μM fucoxanthin induced apoptosis of T24 cells by the abrogation of morta- lin-p53 complex and the reactivation of nuclear mutant- type p53, which also had tumor suppressor function as wild-type p53. All these results demonstrated that the anti- cancer activity of fucoxanthin on T24 cells was associated with cell cycle arrest at Go/G1 phase by up-regulation of p21 at low doses and apoptosis via decrease in the expres- sion level of mortalin, which is a stress regulator and a mem- ber of heat shock protein 70, followed by up-regulation of cleaved caspase-3 at high doses.     

AtKP1, a kinesin-like protein, mainly localizes to mitochondria in Arabidopsis thaliana

Kinesins and kinesin-like proteins （KLPs） constitute a large family of microtubule-based motors that play important roles in many fundamental cellular and developmental processes. To date, a number of kinesins or KLPs have been identified in plants including Arabidopsis thaliana. Here, a polyclonal antibody against AtKP1 （kinesin-like protein 1 in A. thaliana） was raised by injection the expressed AtKP1 specific C-terminal polypeptides in rabbits, and immunoblot analysis was conducted with the affinity-purified anti-AtKP1 antibody. The results indicated that this antibody recognized the AtKP1 fusion proteins expressed in E. coli and proteins of -125 kDa in the soluble fractions of Arabidopsis extracts. The molecular weight was consistent with the calculated molecular weight based on deduced amino acids sequence of AtKP1. To acquire the subcellular localization of the protein, AtKP1 in Arabidopsis root cells was observed by indirect immunofluorescence microscopy. AtKP1 was localized to particle-like organelles in interphase or dividing cells, but not to mitotic microtubule arrays. Relatively more AtKP1 was found in isolated mitochondria fraction on immunoblot of the subcellular fractions. The AtKP1 protein could not be released following a 0.6 M KI washing, indicating that AtKP1 is tightly bind to mitochondria and might function associated with this kind of organelles.