Therapeutic peptides are increasingly gaining popularity for medicinal use in a variety of applications , including tumor vaccines , antimicrobial therapy , and nucleic acid delivery . TfR-binding peptide was identified as both diagnostic and potential therapeutic purposes by biopanning through sequential rounds of negative and positive selection . Several peptides derived from phage-displayed peptide library screenings have been developed into drug candidates and tested in clinical trials, thus validating their peptide-targeting potential . Following this research, the development of new peptide-based cancer therapeutics has been undertaken . It is also known that peptide therapeutics are relatively easily generated using either recombinant or solid-phase chemical synthesis techniques and are generally less expensive than antibody-based therapeutics. In addition, because these peptides have lower molecular weights than proteins there is less steric hindrance and the advantage of improved target accessibility.
In this study we linked two functional domains to produce a novel bifunctional peptide that binds to TfR to cause lytic cell killing. It has been shown that the lytic sequence utilized in this study has higher selectivity than other lytic-type sequences thus far tested  in its discrimination between normal and cancer membranes and is suitable for chimerization with a targeting sequence. Accordingly, the cancer selectivity of TfR-lytic hybrid peptide designed in this study for TfR-expressing cancer cells was confirmed in vitro (Table 1 and Figure 2). Due to its targeting moiety, the hybrid peptide demonstrates selective killing of cancer cells as swiftly as 10 min after treatment (Figure 4A and 4B).
The TfR on normal cells is ubiquitously expressed at low levels and is expressed at greater levels on cells with a high proliferation rate, such as cells of the basal epidermis and intestinal epithelium [3–5]. Activated peripheral blood mononuclear cells express high levels of TfR [34–36]. In malignant tissues, TfR is expressed more abundantly than in normal counterpart tissue . Therefore, TfR could be a relevant target for molecular targeted therapies against tumors.
The growth-inhibitory properties of anti-TfR antibodies have been pursued since the 1980s [38, 39]. Various antibodies targeting TfR have shown different modes of action in different models, including blocking of transferrin binding to the receptor , blocking the internalization of the TfR-transferrin complex , downregulating cell-surface TfR , or causing intracellular degradation of TfR . However, as Ng et al. indicated, all anti-TfR antibodies inhibit cell growth through iron deprivation . Moura et al.  and Ng et al.  also showed that treatment of a neutralizing monoclonal antibody (mAb A24) and anti-TfR-avidin fusion protein (anti-rat TfR IgG3-Av) for 48 h and 96 h respectively, effectively inhibited proliferation of cancer cells. However, in this study, our data demonstrate that TfR-lytic peptide induces cancer cell-death as quickly as 10-15 min after treatment (Figure 4A and 4B). We hypothesize that the cytotoxic mechanism of TfR-lytic peptide is initiated by binding of the TfR-binding moiety of the hybrid peptide to TfR molecules on the cell surface, after which the lytic moiety of the hybrid peptide preferentially disintegrates the cancer cell membrane, induces mitochondrial damage, and triggers apoptotic cell death. Currently it is not clear how these lytic-type peptides induce apoptotic cell death on the cell surface. In this study, we demonstrated that TfR-lytic hybrid peptide induced annexin V-PI- and caspase 3&7-PI-positive cells, resulting in the collapse of mitochondrial membrane potential in cancer cells. Active caspases 3&7 are effector caspases activated by stimulation from mitochondria, cell-surface receptors, and endoplasmic reticulum, and by direct stimulation from stress-inducing molecules. In addition, we performed confocal fluorescence microscopy analysis and assessed the mitochondrial membrane potential by JC-1 staining to show that TfR-lytic peptide causes stimulation of the cell surface, suggesting that these cascades are all activated swiftly and simultaneously. Since TfR-lytic peptide quickly disintegrates the cell membrane and accumulates inside the cell, it is assumed that caspase cascades occur simultaneously when the hybrid peptide is administered. We measured the cytotoxic activity of TfR-lytic hybrid peptide to various cancer cell lines in which the expression levels of TfR are from high to low levels. The fold cytotoxic activation of COLO587 and HuCCT1 by TfR-lytic peptide was not so high, because the expression levels of TfR in these cells were low. The enhancement of cytotoxic activity by TfR-lytic peptide depends on the expression levels of TfR in cell lines, suggesting that TfR-lytic peptide is effectively targeted to cancer cell lines in which TfR is expressed dominantly. In addition, as we showed in Figure 4and additional file 3A, the speed of cancer cell killing by TfR-lytic peptide depends on the expression levels of TfR. We also previously showed that the designed lytic peptide was suitable for the conjugation to exert the enhancement of cytotoxic activity both in vitro and in vivo .
Although it is suggested that peptides are relatively easily inactivated by serum components in the human body, it has been shown that diastereomeric peptides are relatively free from inactivation in serum , and that a lytic diastereomeric peptide administered intravenously reduces tumor growth in an animal model of human prostate cancer without rapid degradation of the peptide in blood at a dose of 9 mg/kg . Also, in our previous study, it was found that EGFR-lytic hybrid peptide targeting epidermal growth factor receptor (EGFR) administrated intravenously reduced the growth of EGFR-expressing tumor with a dose as low as 2 mg/kg . IC50 of TfR-lytic is 5-10 μM in vitro, which is approximately 18.5 to 37 mg/L. Given that a total blood volume of the nude mice of about 20 g (body weight) is 1.5 ml, we expected that TfR-lytic may exert enough antitumor effects at a dose of approximately 2.8 to 5.6 mg/kg. In this in vivo study, TfR-lytic-treated group showed 42% of tumor growth-inhibitory effect at a dose of 3 mg/kg. We demonstrated that this dose coincide with that of expected from in vitro data.
So far, several drug candidates, including TfR antibody 42/6 and TfR-diphtheria immunotoxin, have shown limited antitumor activity without severe side effects in clinical trials [48–50]. We expect that TfR-lytic hybrid peptide may offer new options for TfR-targeted cancer therapies. Standard therapy for malignant gliomas usually includes surgical debulking or biopsy, external beam radiation therapy, and systemic chemotherapy. These treatments are incomplete because some tumor cells are allowed to survive, leading to tumor progression or recurrence. TfR-lytic peptide, like all targeted cytotoxins, offers the possibility of targeting these refractory tumor cells because malignant glioma cells express TfR . Our current in vitro results have shown a clear dependence of the drug on the TfR moiety, suggesting high selectivity for tumor cells and less cytotoxity toward normal cells. This selective targeting ability should provide a large therapeutic window of opportunity for targeting cancer cells over normal cells. Further studies to confirm its efficacy, safety, and immunogenicity will broaden the indications of TfR-lytic hybrid peptide for the future.