Temporin-La

Melanoma is a common malignant skin tumor, which is the only fatal skin tumor at present. Melanoma has a high degree of malignancy and metastasis. The activity of modified Temporin-La (T-La) peptides from bullfrog skin were evaluated for antitumor activity and improved targeting in melanoma cells. The amino acid sequence of T-La was modified, resulting in the antitumor peptide, T-La (FS). T-La and T-La (FS) were coupled to the RGD small molecule polypeptide to form the chimeric peptides RGD-T-La and RGD-T-La (FS), respectively. The secondary structures for the peptides, evaluated using circular dichroism, were found to be α-helical. The structure of T-La was evaluated using bioinformatics. In addition, the antitumor effects of the modified peptide and the targeting of RGD chimeric peptide to the tumor in vivo and in vitro were analyzed. Antitumor activity was measured in vitro using the MTT assay. Tumor cells with high integrin αvβ3 expression were detected using flow cytometry, and tumor cells were screened for sensitivity to RGD-T-La (FS) to establish a tumor model in nude mice. The effects of the peptides on tumor cells were measured using laser confocal microscopy in real-time. The mechanism of the peptide antitumor activity in tumor cells was evaluated with scanning electron microscopy. B16 melanoma cells were the most sensitive to the peptides, for which the cell survival rate was 24.65% for 10 µg/ml RGD-T-La (FS). RGD-La (FS) had a rapid effect on tumor cells. RGD chimeric polypeptides exhibited site-targeting cytotoxic effects in tumor cells. In the B16 melanoma mouse model, the peptides exhibited antitumor effects against early melanoma development and induced tumor apoptosis, possibly by inhibiting VEGF and promoting caspase-3 expression. Overall, the present study provides a scientific basis for the application of small molecule antimicrobial peptides as targeted antitumor agents and lays the foundation for the clinical application of these peptides as antitumor drugs.

How to target cancer cells with high specificity and kill cancer cells with high efficiency remains an urgent demand for anticancer drugs. Temporin-La, which belongs to the family of temporins, presents antitumor activity against many cancer cell lines. We first used a whole bioinformatic analysis method as a platform to identify new anticancer antimicrobial peptides (AMPs). On the basis of these results, we designed a temporin-La analog (temporin-Las) and related constructs containing the Arg-Gly-Asp (RGD) tripeptide, the integrin αvβ3 homing domain (RGD-La and RGD-Las). We detected a link between the net charges and integrin αvβ3 expression of cancer cell lines and the antitumor activities of these peptides. Temporin-La and its synthetic analogs inhibited cancer cell proliferation in a dose-dependent manner. Evidence was provided that the affinity between RGD-Las and tumor cell membranes was stronger than other tested peptides using a pull-down assay. Morphological changes on the cell membrane induced by temporin-La and RDG-Las, respectively, were examined by scanning electron microscopy. Additionally, time-dependent morphological changes were detected by confocal microscopy, where the binding process of RGD-Las to the cell membrane could be monitored. The results indicate that the electrostatic interaction between these cationic peptides and the anionic cell membrane is a major determinant of selective cell killing. Thus, the RGD tripeptide is a valuable ligand motif for tumor targeting, which leads to an increased anticancer efficiency by RGD-Las. These AMP-derived peptides have clinical potential as specifically targeting agents for the treatment of αvβ3 positive tumors.

Malignant melanoma, an increasingly common form of skin cancer, poses a significant threat to public health, especially when the disease progresses past skin lesions to the stage of advanced metastasis. In this work, a new anti-tumor peptide, temporin La (T-La), was selected from a cDNA library generated from bullfrog skin. Two new derivative antitumor peptides, T-La (S) and T-La (FS), were designed by bioinformatics analysis and coupled with the RGD small molecule peptide to create chimeric RGD peptides, (RGD-T-La [S] and RGD-T-La [FS]). Preliminary experiments showed that the new antitumor peptides had significant antitumor effects. After coupling to the chimeric RGD peptide, the targeted treatment of mouse melanoma was significantly improved. Our data demonstrate that the 4 peptides tested herein significantly inhibited the proliferation, migration, and invasion of B16F10 cells; with an increase in polypeptide concentration, the proportion of melanoma cells in the G0/G1 phase decreased or increased significantly, respectively, the reactive oxygen species (ROS) content increased significantly, the mitochondrial membrane potential decreased significantly, and the expression of pro-apoptotic Bax, Caspase-3, and Caspase-9 increased, and anti-apoptotic Bcl-2 decreased significantly. Tyr and MITF genes were significantly downregulated. In conclusion, the use of these new anti-tumor peptides, when combined with a chimeric RGD peptide, may increase ROS levels and decrease mitochondrial membrane potential by inhibiting the activity of mitochondria, thus releasing apoptosis-promoting factors in B16F10 cells. The present study describes a new potential strategy for the application of promising peptides in the treatment of various cancers.