A Novel Mutant of rLj-RGD3 (rLj-112) Suppressed the Proliferation and Metastasis of B16 Cells through the EGFR Signaling Pathway.

Lj-RGD3, which contains three Arg⁻Gly⁻Asp (RGD) motifs, was first identified from the buccal glands of Lampetra japonica and has been shown to suppress the tumor progression in the previous studies. Apart from the three RGD motifs, Lj-RGD3 is also characterized by its high content of histidine in its amino acid sequence. In order to clarify whether the histidine-rich characterization of Lj-RGD3 is also associated with its anti-tumor activity, mutants were designed in which the three RGD motifs (Lj-112), or all histidines (Lj-27) or both (Lj-26) were deleted. Furthermore, a mutant (Lj-42) in which all histidines and three RGD motifs were respectively substituted with alanines and three Ala⁻Gly⁻Asp (AGD) motifs, as well as a mutant (Lj-41) in which all histidines were substituted with alanines was synthesized to avoid alterations in structure which might further cause changes in the peptides' functions. After recombination and purification, recombinant Lj-112 (rLj-112), recombinant Lj-27 (rLj-27), recombinant Lj-41 (rLj-41), and recombinant Lj-RGD3 (rLj-RGD3) exhibited anti-proliferative activity in B16 cells, respectively; while recombinant Lj-26 (rLj-26) and recombinant Lj-42 (rLj-42) did not affect the proliferation of B16 cells significantly. In addition, the anti-proliferative activity of rLj-112 in B16 cells was due to apoptosis. Typical apoptosis features were observed, including chromatin condensation, fragmented DNA, and increased levels of cleaved caspase 3/caspase 7/nuclear enzyme poly (ADP-ribose) polymerase (PARP) in B16 cells. Similar to rLj-RGD3, rLj-112 was also capable of suppressing the migration and invasion of B16 cells by disturbing the F-actin arrangement. After labeling with FITC, rLj-112 was found localized in the cytoplasm of B16 cells, which induced the internalization of epidermal growth factor receptor (EGFR), suggesting that rLj-112 might block the EGFR mediated signaling pathway. Actually, the phosphorylation level of EGFR and its downstream signal molecules including Akt, PI3K, p38, and ERK1/2 was reduced in the rLj-112 treated B16 cells. In vivo, rLj-112 also inhibited the growth, weight, and volume of the tumors in B16 xenografted C57BL/6 mice without reducing their body weight, indicating that rLj-112 might be safe and might be used as an effective anti-tumor drug in the near future.

The anti-tumor effects of the recombinant toxin protein rLj-RGD3 from Lampetra japonica on pancreatic carcinoma Panc-1 cells in nude mice.

Recombinant Lampetra japonica RGD peptide (rLj-RGD3) is a soluble toxin protein with three RGD (Arg-Gly-Asp) motifs and a molecular weight of 13.5kDa. The aim of this study was to investigate the effects and mechanisms of rLj-RGD3 on tumor growth and survival in pancreatic carcinoma Panc-1 cell-bearing mice. A Panc-1 human pancreatic carcinoma-bearing nude mouse model was successfully generated, and the animals were treated with different doses of rLj-RGD3 for 3 weeks. The volume and weight of the subcutaneous tumors, the survival of the nude mice, histopathological changes, the intratumoral MVD, the number of apoptotic Panc-1 cells, and apoptosis-related proteins and gene expressions were determined. rLj-RGD3 significantly decreased the tumor volumes and weights, and the maximum tumor volume and weight IR values were 53.2% (p<0.001) and 55.9% (p<0.001), respectively. The life expectancy of Panc-1-bearing nude mice treated with rLj-RGD3 was increased by 56.3% (p<0.001). Meanwhile, rLj-RGD3 promoted the expression of Bax, caspase-3, and caspase-9 and inhibited Bcl-2 and VEGF expression. In addition, rLj-RGD3 did not change FAK, PI3K and Akt expression, but p-FAK, p-PI3K and p-Akt, levels were down-regulated. These results show that rLj-RGD3 induced potent anti-tumor activity in vivo and suppressed the growth of transplanted Panc-1 cells in a nude mouse model, implying that rLj-RGD3 may serve as a potent clinical therapeutic agent for human pancreatic carcinoma.

[Anti-angiogenic activities of Lj-RGD3 toxin protein from Lampetra japonica and its mutation protein Lj-112 without RGD motifs].

Arg-Gly-Asp (RGD)-toxin protein Lj-RGD3 of Lampetra japonica shares homologous with a Histidine-rich glycoprotein (HRG), and both RGD-toxin protein and HRG have antiangiogenic activities with different targets. To study the relationship between the function and the structure of Lj-RGD3, we studied the anti-angiogenic characteristics of both Lj-RGD3 and the mutation named Lj-112 of which three RGD motifs of Lj-RGD3 were deleted. We synthesized the gene of Lj-112, constructed it to the plasmid pET23b, and expressed the recombinant proteins in Escherichia coli BL21. Both recombinant proteins with the C-terminal his-tag were 15 kDa soluble proteins. Then we purified rLj-RGD3 and rLj-112 using the His-Bind affinity chromatography. To examine the effect of both proteins on bFGF-induced proliferation of ECV304 cell, we carried out the 3-(4,5)-dimethylthiahiazo (-z-yl)-3,5-di-phenytetrazoliumromide (MTT) assays. For cell migration and invasion assays, we used Transwell containing insert filter and Matrigel to imitate the in vivo environment. To examine whether both proteins were capable of interrupting the angiogenesis in vivo, we used the chick chicken embryonic chorioallantoic membrane (CAM) as an angiogenesis model. We used Integrin-linked kinase1 (ILK1) ELISA method to study functionary mechanisms of rLj-RGD3 and rLj-112. Both rLj-RGD3 and rLj-112 inhibited bFGF-induced proliferation of ECV304 cells in a dose-dependent manner with IC50 at 0.889 micromol/L and 0.160 micromol/L, respectively. The results of migration and invasion assays revealed that both rLj-RGD3 and rLj-112 showed significant inhibition on bFGF induced migration and invasion of ECV304; and rLj-112 was more active than rLj-RGD3. The result of CAM angiogenesis assay demonstrated that both proteins inhibited the angiogenesis in chick CAM, and rLj-112 was more active than rLj-RGD3. ELISA assay of ILK1 showed that both rLj-RGD3 and rLj-112 down-regulated ILK1 expression of ECV304 cell. The fact of rLj-112 was more active than rLj-RGD3 on anti-angiogenesis indicate that rLj-112 was likely with histidine-rich glycoprotein (HRG), and the factor of sequence homologous between rLj-RGD3 and HRG cannot enhance antiangiogenic activities of rLj-RGD3, the signal pathway of anti-angiogenesis of rLj-RGD3 and rLj-112 are differently.

[Expression and bioactivity effects to Hela of recombinant toxin protein rLj-RGD3 from Lampetra japonica].

Lj-RGD3 was a toxin from the saliva gland of Lampetra japonica. To study the anti-tumor function of rLj-RGD3 and confirm its biological status and significance, we extracted total RNA from the saliva gland and amplified the cDNA of Lj-RGD3 by RT-PCR. The cDNA of Lj-RGD3 was 357 bp long and encoded a polypeptide composed of 118 amino acids including 2 cysteines, 17 histidines and 3 RGD (Arg-Gly-Asp) motifs. We cloned the cDNA into the plasmid pET23b, and expressed the recombinant protein rLj-RGD3 in Escherichia coli BL21. Fusion rLj-RGD3 with the C-terminal his-tag was a 15 kD soluble protein. Using the His-Bind affinity chromatography, we purified rLj-RGD3. Furthermore, we determined the biological activities of rLj-RGD3. To examine the ability of rLj-RGD3 inhibiting Hela cells proliferation, we used MTT assay. The results showed that, rLj-RGD3 inhibited bFGF induced proliferation of Hela cells in a dose-dependent manner, the IC50 value was 2.6 micromol/L. Hoechst staining assay revealed that, the nuclei of the cells treated with rLj-RGD3 were stained much brighter than that of untreated cells due to chromatin condensation. Furthermore, the DNA ladder patterns from the cells treated with rLj-RGD3 were also observed. These results demonstrated that rLj-RGD3 could induce apoptosis of Hela cells. Cell adhesion, migration and invasion are critical processes in tumor metastasis. rLj-RGD3 significantly inhibited adhesion of Hela cells to vironectin in a dose-dependent manner. In order to determine the effect of rLj-RGD3 on Hela cells migration toward bFGF, we used Transwell containing insert filter. rLj-RGD3 showed a significant inhibition on Hela cells migration, the inhibition rate was 60%. In the invasion assay, the Matrigel and Transwell were used to imitate environment in vivo. The results of invasion assay revealed that, rLj-RGD3 significantly inhibited bFGF induced invasion of Hela cells. Taken together, these results revealed that rLj-RGD3 had typical functions of RGD toxin protein and will be valuable in developing anti-tumor recombinant medicine.

Molecular cloning of grammistins, peptide toxins from the soapfish Pogonoperca punctata, by hemolytic screening of a cDNA library.

A novel method, based on the hemolytic screening of a cDNA phage library, was developed to isolate cDNAs encoding grammistins (antibacterial peptide toxins) of the soapfish Pogonoperca punctata. As a result, cDNAs encoding six grammistins were isolated and elucidated for their nucleotide sequences. In common with the grammistins, the precursor protein is composed of a highly conserved signal peptide, a considerably conserved propeptide that is characterized to contain a pair of basic residues (Lys-Arg) at plural positions including the C-terminus and one copy of a mature peptide. This precursor organization is similar to those of dermaseptins, antibacterial peptides from the frog skin.

Venom evolution widespread in fishes: a phylogenetic road map for the bioprospecting of piscine venoms.

Knowledge of evolutionary relationships or phylogeny allows for effective predictions about the unstudied characteristics of species. These include the presence and biological activity of an organism's venoms. To date, most venom bioprospecting has focused on snakes, resulting in six stroke and cancer treatment drugs that are nearing U.S. Food and Drug Administration review. Fishes, however, with thousands of venoms, represent an untapped resource of natural products. The first step involved in the efficient bioprospecting of these compounds is a phylogeny of venomous fishes. Here, we show the results of such an analysis and provide the first explicit suborder-level phylogeny for spiny-rayed fishes. The results, based on approximately 1.1 million aligned base pairs, suggest that, in contrast to previous estimates of 200 venomous fishes, >1,200 fishes in 12 clades should be presumed venomous. This assertion was corroborated by a detailed anatomical study examining potentially venomous structures in >100 species. The results of these studies not only alter our view of the diversity of venomous fishes, now representing >50% of venomous vertebrates, but also provide the predictive phylogeny or "road map" for the efficient search for potential pharmacological agents or physiological tools from the unexplored fish venoms.

Complete amino-acid sequence of the beta-subunit of VTX from venom of the stonefish (Synanceia verrucosa) as identified from cDNA cloning experiments.

A cDNA encoding a subunit of the verrucotoxin (VTX) has been identified from a cDNA library derived from stonefish venom glands. It encodes a polypeptide of 708 amino-acid residues, followed by a 3'-untranslated region of 895 bp long. The ORF contains the complete mature sequence of the beta-subunit of the VTX, as inferred from both the presence of an identical N-terminus sequence and 96% homology among the 506 amino terminus residues found in the partial sequence of the beta-subunit of the stonustoxin from Synanceia horrida (Ghadessy, F.J., Jeyaseelan, K., Chung, M.C.M., Khoo, H.E., and Yuen, R. (1994) Toxicon 32, 1684-1688). Upstream the mature sequence, we noticed the presence of an incomplete peptide of a 13 amino acids, whose unusual primary structure supports the idea of the existence of a propeptide and/or of a new secretion signal.