Oligopeptides derived from autophosphorylation sites of EGF receptor suppress EGF-stimulated responses in human lung carcinoma A549 cells.

Epidermal growth factor (EGF) receptor plays a crucial role in the biology of human cancer, and is a highly appropriate target for anticancer agents. We have previously designed oligopeptides containing the amino acid sequences around autophosphorylation sites of EGF receptor to identify a specific inhibitor of this receptor. We found that Ac-ENAEYLR-NH(2) and Ac-NYQQN-NH(2) suppressed phosphorylation of purified EGF receptor in a non-ATP-competitive manner whereas Ac-QNAQYLR-NH(2) and Ac-DYQQD-NH(2) caused inhibition in an ATP-competitive manner. The aim of this study was to observe the effects of these peptides on the proliferation, cell death, and apoptosis of human lung carcinoma A549 cells. To facilitate transfer of these inhibitory peptides into A549 cells, the cell-penetrating peptide, human immunodeficiency virus type 1-transactivator of transcription (Tat), was linked to the peptides. When A549 cells were treated with each Tat-conjugated peptide, the peptides penetrated the cells and EGF-stimulated tyrosine phosphorylation of EGF receptor was significantly suppressed. These Tat-conjugated peptides played a suppressive role in EGF-stimulated A549 cell responses. In particular, Tat-epsilon-aminocaproic acid (acp)-ENAEYLR-NH(2) significantly inhibited proliferation and showed cytotoxicity, while Tat-acp-NYQQN-NH(2) and Tat-acp-DYQQD-NH(2) suppressed the anti-apoptotic effect of EGF. In addition, we found that Tat-acp-ENAEYLR-NH(2) also inhibited the phosphorylation of epidermal growth factor receptor 2 (ErbB2) as well as EGF receptor in A549 cells. In conclusion, membrane-permeable synthetic peptides derived from EGF receptor autophosphorylation sites have the potential to suppress EGF receptor function in A549 cells and to be developed into novel and useful agents for cancer therapy.

Effects of TrkA inhibitory peptide on cancer-induced pain in a mouse melanoma model.

PURPOSE: Tropomyosin receptor kinase (Trk) A, a high-affinity receptor of nerve growth factor, is a therapeutic target for both noxious and neuropathic pain. The present study examined the effects of an inhibitory peptide of Trk activity (IPTRK) 3 that inhibits TrkA activity on cancer-induced pain in a mouse melanoma model. METHODS: The hind paws of mice were inoculated with B16-F1 mouse melanoma cells on day 0. We administered IPTRK3 (20 mg/kg i.p.) repetitively on days 5, 6, 7, 8, and 9, and evaluated pain-related behaviors on days 0, 5, 10, 15, and 20 after tumor inoculation. RESULTS: Following inoculation, mice demonstrated mechanical allodynia and thermal hyperalgesia with an increased number of flinches, and paw volume increased gradually. However, an intraperitoneal injection of IPTRK3 significantly inhibited mechanical allodynia on day 15 and suppressed the number of flinches on day 20. The increased paw volume was significantly suppressed on day 20 after tumor inoculation. IPTRK3, however, showed no significant effect on thermal hyperalgesia. CONCLUSIONS: These results suggest that TrkA inhibitory peptide likely suppress melanoma-induced pain with concomitant reduction in the increased paw volume in a mouse skin cancer pain model.

A synthetic cell-penetrating peptide antagonizing TrkA function suppresses neuropathic pain in mice.

Nerve growth factor (NGF) and its high-affinity receptor, TrkA, are one of the targets in the production of new drugs for the treatment of neuropathic pain. NGF contributes to both the initiation and maintenance of sensory abnormalities after peripheral nerve injury. This study examined the effects of IPTRK3, a new synthetic cell-penetrating peptide that antagonizes TrkA function, on neuropathic pain in mice. Partial sciatic nerve ligation (PSNL) was used to generate neuropathic pain, and we injected IPTRK3 (2 or 10 mg/kg) intraperitoneally on day 7 after PSNL. Effects of the peptide on hyperalgesia, allodynia, and expression of Fos in the spinal cord were examined. Single administration of the peptide on day 7 significantly suppressed both thermal hyperalgesia and mechanical allodynia. Gentle touch stimuli-evoked Fos expression in the lumbar spinal cord was also significantly reduced. Intraperitoneal injection of a cell-penetrating peptide antagonizing TrkA function appears effective for treatment of neuropathic pain in a mouse pain model.

Local administration of a synthetic cell-penetrating peptide antagonizing TrkA function suppresses inflammatory pain in rats.

Novel agents that inhibit nerve growth factor signaling are required for the treatment of inflammatory pain. The present study investigated the effect of local administration of inhibitory peptide of TrkA (IPTRK3), a synthetic cell-penetrating peptide that antagonizes TrkA function, in complete Freund's adjuvant (CFA)-induced hyperalgesia in rats. Three hours after subcutaneous injection of CFA into the plantar surface of the rat's left hind paw, 10 mM IPTRK3 was injected at the same site. Thermal and mechanical hyperalgesia were tested in the ipsilateral hind paw until 7 days after CFA injection. The ipsilateral dorsal root ganglion (DRG) was dissected out for immunohistochemical analysis of transient receptor potential vanilloid subfamily member 1 (TRPV1) channels and TrkA. Local injection of this peptide significantly suppressed both thermal and mechanical hyperalgesia produced by CFA and also significantly reduced TRPV1 expression at the DRG. These results suggest that local administration of IPTRK3 is likely effective in the treatment of inflammatory pain in rats.

Effect of synthetic cell-penetrating peptides on TrkA activity in PC12 cells.

As TrkA, a high-affinity receptor of nerve growth factor (NGF), is a potential target for relieving uncontrolled inflammatory pain, an effective inhibitor of TrkA has been required for pain management. To identify a specific inhibitor of TrkA activity, we designed cell-penetrating peptides combined with amino-acid sequences in the activation loop of TrkA to antagonize tyrosine kinase activity. To select a peptide inhibiting TrkA activity, we examined the effect of cell-penetrating peptides on tyrosine kinase activity of recombinant TrkA in vitro and studied their effects on NGF-stimulated neurite outgrowth and protein phosphorylation in PC12 cells. Thereafter we investigated the effect of the selected peptide on NGF-stimulated TrkA activity and the expression of transient receptor potential channel 1 in PC12 cells. The selected peptide inhibited TrkA activity, but did not inhibit tyrosine kinase activities of other receptor-type tyrosine kinases in vitro. It also suppressed NGF-stimulated responses in PC12 cells. The selected synthetic cell-penetrating peptide antagonizing TrkA function would be a candidate for inflammatory pain therapy.