Synergetic cytotoxic activity toward breast cancer cells enhanced by the combination of Antp-TPR hybrid peptide targeting Hsp90 and Hsp70-targeted peptide.

BACKGROUND: Heat shock protein (Hsp) 90 and Hsp70 are indispensable for cell survival under conditions of stress. They bind to client proteins to assist in protein stabilization, translocation of polypeptides across the cell membrane, and recovery of proteins from aggregates in the cell. Therefore, these proteins have recently emerged as important targets in the treatment of cancer. We previously reported that the newly designed Antp-TPR hybrid peptide targeting Hsp90 induced cytotoxic activity to cancer cells both in vitro and in vivo. METHODS: To further improve the cytotoxic activity of Antp-TPR toward cancer cells, we investigated the effect of a Hsp70-targeted peptide, which was made cell-permeable by adding the polyarginine with a linker sequence, on the cytotoxic activity of Antp-TPR in breast cancer cell lines. RESULTS: It was revealed that Antp-TPR in the presence of a Hsp70-targeted peptide induced effective cytotoxic activity toward breast cancer cells through the descrease of Hsp90 client proteins such as p53, Akt, and cRaf. Moreover, the combined treatment with these peptides did not induce the up-regulation of Hsp70 protein, as determined by western blotting, a promoter assay using a luminometer, and single-cell level imaging with the LV200 system, although a small-molecule inhibitor of Hsp90, 17-allylamino-demethoxygeldanamycin (17-AAG), did induce the up-regulation of this protein. We also found that treatment with Antp-TPR, Hsp70-targeted peptide, or a combination of the two did not induce an increase in the glutathione concentrations in the cancer cells. CONCLUSION: These findings suggest that targeting both Hsp90 and Hsp70 with Antp-TPR and Hsp70-targeted peptide is an attractive approach for selective cancer cell killing that might provide potent and selective therapeutic options for the treatment of cancer.

Localization of the anti-cancer peptide EGFR-lytic hybrid peptide in human pancreatic cancer BxPC-3 cells by immunocytochemistry.

Cationic lytic-type peptides have been studied for clinical application in various infections and cancers, but their functional cellular mechanisms remain unclear. We generated anti-cancer epithelial growth factor receptor (EGFR)-lytic hybrid peptide, a 32-amino-acid peptide composed of an EGFR-binding sequence and lytic sequence. In this study, we investigated the distribution of EGFR-lytic hybrid peptide in BxPC-3 human pancreatic cancer cells by an immunocytochemical (ICC) method. Distribution of EGFR protein expression was unchanged after treatment with EGFR-lytic peptide compared with non-treated cells. In confocal laser scanning microscopy, immunostaining of EGFR-lytic peptide was observed in the cytoplasm, mostly in the form of granules. Some staining was also localized on the mitochondrial membrane. At the ultrastructure level, cells treated with EGFR-lytic peptide had a low electron density, disappearance of microvilli, and swollen mitochondria. Fragments of cell membrane were also observed in the proximity of the membrane. In immunoelectron microscopy, EGFR-lytic peptide was observed in the cell membrane and cytoplasm. A number of granules were considered swollen mitochondria. Activation of the caspase pathway as a result of mitochondrial dysfunction was also examined to determine the cytotoxic activity of EGFR-lytic peptide; however, no effect on cell death after EGFR-lytic treatment was observed, and moreover, apoptosis was not found to play a critical role in the cell death mechanism. These results suggest that EGFR-lytic peptide is localized on cell and mitochondrial membranes, with disintegration of the cell membrane contributing mainly to cell death.

Molecular mechanism of cytotoxicity induced by Hsp90-targeted Antp-TPR hybrid peptide in glioblastoma cells.

BACKGROUND: Heat-shock protein 90 (Hsp90) is vital to cell survival under conditions of stress, and binds client proteins to assist in protein stabilization, translocation of polypeptides across cell membranes, and recovery of proteins from aggregates. Therefore, Hsp90 has emerged as an important target for the treatment of cancer. We previously reported that novel Antp-TPR hybrid peptide, which can inhibit the interaction of Hsp90 with the TPR2A domain of Hop, induces selective cytotoxic activity to discriminate between normal and cancer cells both in vitro and in vivo. RESULTS: In this study, we investigated the functional cancer-cell killing mechanism of Antp-TPR hybrid peptide in glioblastoma (GB) cell lines. It was demonstrated that Antp-TPR peptide induced effective cytotoxic activity in GB cells through the loss of Hsp90 client proteins such as p53, Akt, CDK4, and cRaf. Antp-TPR also did not induce the up-regulation of Hsp70 and Hsp90 proteins, although a small-molecule inhibitor of Hsp90, 17-AAG, induced the up-regulation of these proteins. It was also found that Antp-TPR peptide increased the endoplasmic reticulum unfolded protein response, and the cytotoxic activity of this hybrid peptide to GB cells in the endoplasmic reticulum stress condition. CONCLUSION: These results show that targeting of Hsp90 by Antp-TPR could be an attractive approach to selective cancer-cell killing because no other Hsp90-targeted compounds show selective cytotoxic activity. Antp-TPR might provide potent and selective therapeutic options for the treatment of cancer.

A single replacement of histidine to arginine in EGFR-lytic hybrid peptide demonstrates the improved anticancer activity.

We previously reported that novel targeted "hybrid peptide" in which epidermal growth factor receptor (EGFR) binding peptide was conjugated with lytic-type peptide had selective cytotoxic activity to EGFR expressing cancer cell lines, and in vivo analysis revealed that this EGFR-lytic peptide displayed significant antitumor activity in a xenograft model of human breast cancer which was resistant to tyrosine kinase inhibitor drugs. As an attempt to improve the selective anticancer activity of EGFR-lytic peptide, we modified the EGFR-binding peptide through introducing the mutation of amino acid according to biophysical analysis by biomolecular interaction and circular dichroism (CD) spectra. When cytotoxic activity of EGFR-lytic or EGFR(2R)-lytic hybrid peptides was investigated in various human cancer and normal cell lines, it was demonstrated that EGFR(2R)-lytic, in which second histidine (H) of EGFR-binding peptide was replaced to arginine (R) had 1.2-1.9-fold higher cytotoxic activity than that of original EGFR-lytic peptide. In vivo analysis also revealed that this modified peptide displayed significant antitumor activity at as low as 1 mg/kg dosage. These results suggest that mutated arginine on EGFR-lytic peptide produces higher binding ability to EGFR on cancer cells, and thereby the improved anticancer activity.

Semaphorin 3A lytic hybrid peptide binding to neuropilin-1 as a novel anti-cancer agent in pancreatic cancer.

We previously reported that novel targeted "hybrid peptide" in which epidermal growth factor receptor (EGFR) binding peptide was conjugated with lytic-type peptide had selective cytotoxic activity to EGFR expressing cancer cells. In this study, we have generated a novel type hybrid peptide, semaphorin 3A lytic (Sema3A-lytic), which is composed of two functional amino acid domains: a sequence derived from Sema3A that binds to neuropilin-1 (NRP1) and a cytotoxic lytic peptide. We found that this hybrid peptide had cytotoxic activity against NRP1-positive pancreatic cancer cell lines such as BxPC-3 and Panc-1, whereas the peptide did not affect the viability of normal cells in vitro. It was also found by affinity analysis that Sema3A peptide binds to NRP1, and two arginines (372R and 377R) in Sema3A peptide are involved in the interaction with NRP1 protein. In addition, confocal microscopy analysis revealed that Sema3A-lytic peptide could not penetrate normal cells regardless of the presence of NRP1 mRNA, suggesting that the ability of Sema3A-lytic peptide to concentrate adjacent to the cell membrane by binding to NRP1 with the target-binding moiety contributes to its selective cytotoxic activity. These results indicate that Sema3A-lytic hybrid peptide would be a possible anti-cancer agent for treatment of human pancreatic cancer.

Designed hybrid TPR peptide targeting Hsp90 as a novel anticancer agent.

BACKGROUND: Despite an ever-improving understanding of the molecular biology of cancer, the treatment of most cancers has not changed dramatically in the past three decades and drugs that do not discriminate between tumor cells and normal tissues remain the mainstays of anticancer therapy. Since Hsp90 is typically involved in cell proliferation and survival, this is thought to play a key role in cancer, and Hsp90 has attracted considerable interest in recent years as a potential therapeutic target. METHODS: We focused on the interaction of Hsp90 with its cofactor protein p60/Hop, and engineered a cell-permeable peptidomimetic, termed "hybrid Antp-TPR peptide", modeled on the binding interface between the molecular chaperone Hsp90 and the TPR2A domain of Hop. RESULTS: It was demonstrated that this designed hybrid Antp-TPR peptide inhibited the interaction of Hsp90 with the TPR2A domain, inducing cell death of breast, pancreatic, renal, lung, prostate, and gastric cancer cell lines in vitro. In contrast, Antp-TPR peptide did not affect the viability of normal cells. Moreover, analysis in vivo revealed that Antp-TPR peptide displayed a significant antitumor activity in a xenograft model of human pancreatic cancer in mice. CONCLUSION: These results indicate that Antp-TPR peptide would provide a potent and selective anticancer therapy to cancer patients.

A novel transferrin receptor-targeted hybrid peptide disintegrates cancer cell membrane to induce rapid killing of cancer cells.

BACKGROUND: Transferrin receptor (TfR) is a cell membrane-associated glycoprotein involved in the cellular uptake of iron and the regulation of cell growth. Recent studies have shown the elevated expression levels of TfR on cancer cells compared with normal cells. The elevated expression levels of this receptor in malignancies, which is the accessible extracellular protein, can be a fascinating target for the treatment of cancer. We have recently designed novel type of immunotoxin, termed "hybrid peptide", which is chemically synthesized and is composed of target-binding peptide and lytic peptide containing cationic-rich amino acids components that disintegrates the cell membrane for the cancer cell killing. The lytic peptide is newly designed to induce rapid killing of cancer cells due to conformational change. In this study, we designed TfR binding peptide connected with this novel lytic peptide and assessed the cytotoxic activity in vitro and in vivo. METHODS: In vitro: We assessed the cytotoxicity of TfR-lytic hybrid peptide for 12 cancer and 2 normal cell lines. The specificity for TfR is demonstrated by competitive assay using TfR antibody and siRNA. In addition, we performed analysis of confocal fluorescence microscopy and apoptosis assay by Annexin-V binding, caspase activity, and JC-1 staining to assess the change in mitochondria membrane potential. In vivo: TfR-lytic was administered intravenously in an athymic mice model with MDA-MB-231 cells. After three weeks tumor sections were histologically analyzed. RESULTS: The TfR-lytic hybrid peptide showed cytotoxic activity in 12 cancer cell lines, with IC(50) values as low as 4.0-9.3 µM. Normal cells were less sensitive to this molecule, with IC(50) values > 50 µM. Competition assay using TfR antibody and knockdown of this receptor by siRNA confirmed the specificity of the TfR-lytic hybrid peptide. In addition, it was revealed that this molecule can disintegrate the cell membrane of T47D cancer cells just in 10 min, to effectively kill these cells and induce approximately 80% apoptotic cell death but not in normal cells. The intravenous administration of TfR-lytic peptide in the athymic mice model significantly inhibited tumor progression. CONCLUSIONS: TfR-lytic peptide might provide a potent and selective anticancer therapy for patients.

Characterization of antilytic peptide antibody: application for the detection of lytic-based hybrid peptide in serum samples.

We previously reported that a novel targeted drug termed hybrid epidermal growth factor receptor (EGFR)-lytic peptide, made by chemical conjugation of targeted binding peptide and cell-killing, lytic-peptide components, has selective cytotoxic activity that allows it to discriminate between normal and cancer cells. In addition, in vivo analysis revealed that this hybrid peptide displays significant antitumor activity in a xenograft model of human breast and pancreatic cancer in mice. Here, we characterized antilytic peptide antibody, which was raised from rabbit serum using the antigen of lytic peptide conjugated with keyhole limpet hemocyanin. It was found that antilytic peptide antibody is specific to the lytic peptide as assessed by both ELISA and surface plasmon resonance analysis and can also bind to EGFR-lytic peptide. Epitope mapping analysis using Biacore showed that two successive lysine regions in the lytic-peptide sequence are significant for recognition by this antibody. In addition, it was shown that this antibody can detect lytic-based hybrid peptide in serum samples from mouse blood and also in cultured breast cancer MDA-MB-231 cell samples by immunocytochemical staining experiments. It was found that the maximum concentrations of this peptide in serum were reached within 15-30 min of i.v. administration of EGFR-lytic peptide to mice. These results indicate that this antibody will be a useful tool for the detection of lytic-based peptides to investigate their in vivo stability and pharmacokinetics.

Antedrug budesonide by intrapulmonary treatment attenuates bleomycin-induced lung injury in rats with minimal systemic adverse effects.

Corticosteroids are routinely used in patients with pulmonary fibrosis, yet they have several adverse effects. To improve this situation, we used an animal model of pulmonary injury and early fibrosis and investigated whether the combination of an intrapulmonary inhalation device with antedrug budesonide (BUD) administered to the lung had greater efficacy and fewer systemic adverse effects compared to long-acting dexamethasone (DEX). BUD or DEX was administrated either intrapulmonary or intravenously to bleomycin-treated rats. Anti-inflammatory and antifibrotic effects were evaluated according to inflammatory cell count, total protein concentration and soluble collagen concentration in bronchoalveolar lavage fluid. The systemic immunosuppressive effects were also assessed by measuring body, spleen and thymus weight. BUD and DEX were compared with respect to their pharmacokinetic profiles in plasma and lung. Intrapulmonary treatment of BUD attenuates various inflammatory and early fibrotic indices with minimal systemic adverse effects compared with DEX. The area under the curve (AUC) of BUD by intrapulmonary spray was 6.6-fold higher than the AUC of DEX in the lung. This study suggests that antedrug BUD by intrapulmonary treatment has local anti-inflammatory and antifibrotic effects with minimal systemic adverse effects.

Oral administration of ferulic acid or ethyl ferulate attenuates retinal damage in sodium iodate-induced retinal degeneration mice.

Epidemiological studies indicate that the daily intake of antioxidants from a traditional Asian diet reduces the risk of developing age-related macular degeneration. Many of the phytochemicals that are abundant in whole grains exhibit a wide variety of biological activity such as antioxidant, anti-inflammatory, and neuroprotective effects. Ferulic acid (FA) is a phenolic acid found in vegetables and grains that has therapeutic potential for diabetes mellitus, Alzheimer's disease, and other diseases. We investigated the retinal protective effect of FA in a sodium iodate (NaIO3)-induced model of retinal degeneration. In a human retinal pigment epithelial cell line, FA attenuated H2O2-induced injury and lipopolysaccharide- or 7-ketocholesterol-induced inflammation. In mice, the oral administration of FA or its analog, ethyl ferulate, attenuated the morphological and functional features of NaIO3-induced retinal degeneration according to optical coherence tomography and electroretinography. Our results demonstrate that the oral administration of FA provides protective effects to the retina, suggesting that the intake of FA as a daily supplement or daily healthy diet containing rich vegetables and whole grains may prevent age-related macular degeneration.

Use of telomelysin (OBP-301) in mouse xenografts of human head and neck cancer.

We previously reported that telomerase-specific replication-component adenovirous, Telomelysin (OBP-301) has cytotoxic activity to the YCUT892, KCCT873, KCCT891, KCCL871, YCUM862, HN12, and KCCOR891 cell lines in vitro, and investigated the association between cytotoxic activity and adenoviral receptor expression. In this study, we evaluated the most appropriate way to administer telomelysin (OBP-301) in the treatment of squamous cell carcinoma of the head and neck (SCCHN), and assessed the effect of OBP-301 in large subcutaneous KCCT873 human SCCHN tumors in immunodeficient mice. We also compared antitumor responses following three intratumoral (i.t.) injections of OBP-301 given daily, every 2 days or weekly. To investigate the mechanism of the antitumor effect, we evaluated cellular infiltration in treated tumors. OBP-301 showed remarkable antitumor activity against large KCCT873 tumors, and three treatment schedules produced similar antitumor effects. The weekly regimen also significantly reduced the growth of large tumors. Immunochemistry revealed that macrophages, but not natural killer cells, were responsible for tumor regression. A regimen of three weekly injections of OBP-301 has remarkable antitumor effects against large KCCT873 tumors. These results may provide a new platform for treating patients with localized SCCHN.

A novel interleukin-13 receptor alpha 2-targeted hybrid peptide for effective glioblastoma therapy.

We previously designed and reported a novel class of drugs, namely hybrid peptides, which are chemically synthesized and composed of a targeted binding peptide and a lytic-type peptide containing cationic amino acid residues that cause cancer cell death. In the present study, we screened for peptides that bind to interleukin-13 receptor alpha 2 (IL-13Rα2) by using a T7 random peptide phage display library system and isolated several positive phage clones. The A2b11 peptide, which was one of the positive clones, was shown to bind to IL-13Rα2 protein by Biacore analysis and a binding assay using glioblastoma (GB) cell lines. This peptide was linked with a lytic peptide containing a linker sequence to form the IL-13Rα2-lytic hybrid peptide. The IL-13Rα2-lytic hybrid peptide showed cytotoxic activity against GB cell lines in vitro. The IL-13Rα2-lytic hybrid peptide also affected Akt and Erk1/2 activation following treatment with interleukin-13 and induced rapid ATP dynamics in GB cells. Anti-tumor activity of the IL-13Rα2-lytic hybrid peptide was observed in vivo after intratumoral injection in a mouse xenograft model of human GB cells. These results suggest that the IL-13Rα2-lytic hybrid peptide might be a potent therapeutic option for patients with GB.

Bile Acid as an Effective Absorption Enhancer for Oral Delivery of Epidermal Growth Factor Receptor-Targeted Hybrid Peptide.

The aim of this study was to improve the oral absorption of epidermal growth factor receptor-targeted hybrid peptide using bile acid as an absorption enhancer. The oral formulation of this peptide was formed through electrostatic interactions between the cationic peptide and anionic bile acid. Comparative studies of in vitro cell permeability and in vivo antitumor effects of peptide and peptide/bile acid complex were performed in Caco-2 cells and in a xenograft mouse model of human gastric cancer. The in vitro permeability of peptide/bile acid complex across Caco-2 cell monolayers was significantly enhanced to about 5.0-fold over those of peptide alone. Furthermore, in vivo mouse xenograft model treated with peptide/bile acid complex showed a 1.6-fold reduction in the mean tumor volume as compared with the peptide alone. A preliminary safety evaluation of blood cells counts, liver enzyme levels, and histopathology of gastrointestinal tissues and main organs showed that the peptide/bile acid complex did not induce any acute toxicity. These results suggest that bile acid is an effective absorption enhancer for improving the oral bioavailability and bioactivity of epidermal growth factor receptor-targeted hybrid peptide.

Potent anti-tumor effects of EGFR-targeted hybrid peptide on mice bearing liver metastases.

In this study, we investigated the therapeutic efficacy of EGFR2R-lytic hybrid peptide for the treatment of liver metastasis from colon carcinoma. The cytotoxic activity of the hybrid peptide against luciferase-expressing human colon cancer (HCT-116-luc) cells was determined by the WST-8 assay. The experimental mouse model of liver metastases was generated by splenic injection of HCT-116-luc cells. The hybrid peptide was intravenously injected into mice the day after cell implantation at a dose of 5 mg/kg and this was repeated on alternate days for a total of 7 doses. Saline-treated mice were used as controls. Tumor growth and therapeutic responses were monitored by an IVIS imaging system. It was shown that the hybrid peptide exhibited potent cytotoxic activity against HCT-116-luc cells and the liver metastases were significantly reduced after intravenous injections of hybrid peptide compared with controls. Furthermore, Kaplan­Meier analysis showed that hybrid peptide-treated mice had significantly longer survival than controls. In addition, bright-field and ex vivo imaging of liver tissue revealed that mice treated with the hybrid peptide had significantly fewer tumors compared with controls. These results demonstrated that the EGFR2R-lytic hybrid peptide is a potential treatment option for patients with colorectal cancer metastases in the liver.

Novel EGFR-targeted strategy with hybrid peptide against oesophageal squamous cell carcinoma.

Epidermal growth factor receptor (EGFR) is a key molecule in the pathophysiology of oesophageal squamous cell carcinoma (OSCC). However, EGFR-targeted agents such as anti-EGFR antibody or tyrosine kinase inhibitors for OSCC have not demonstrated any clinical benefits. Recently, a novel chemotherapeutic agent, EGFR(2R)-lytic hybrid peptide, a composite of EGFR-binding peptide and lytic peptide fragments, has been shown to exhibit a potent anti-tumour effect against cancers that express high EGFR levels. In this study, we investigated the validity of employing EGFR(2R)-lytic hybrid peptide against OSCC cells both in vitro and in vivo. Additionally, the toxicity of this peptide was assessed in mice. We found high EGFR expression levels on the cell surface of OSCC cells, and the EGFR-binding peptide fragment showed high affinity for OSCC cells. A potent cytotoxic effect was induced within 30 minutes by the exposure of OSCC cells to EGFR(2R)-lytic hybrid peptide. Furthermore, EGFR(2R)-lytic hybrid peptide markedly suppressed the tumour growth of OSCC cells in a xenograft model. Moreover, it did not cause any identifiable adverse effects in mice. Taken together, EGFR(2R)-lytic hybrid peptide was shown to be a valid therapeutic agent against OSCC, providing a crucial rationale regarding novel EGFR-targeted therapies against OSCC.

Enhancement of anti-tumor activity of hybrid peptide in conjugation with carboxymethyl dextran via disulfide linkers.

To improve the anti-tumor activity of EGFR2R-lytic hybrid peptide, we prepared peptide-modified dextran conjugates with the disulfide bonds between thiolated carboxymethyl dextran (CMD-Cys) and cysteine-conjugated peptide (EGFR2R-lytic-Cys). In vitro release studies showed that the peptide was released from the CMD-s-s-peptide conjugate in a concentration-dependent manner in the presence of glutathione (GSH, 2µM-2mM). The CMD-s-s-peptide conjugate exhibited a similar cytotoxic activity with free peptide alone against human pancreatic cancer BxPC-3 cells in vitro. Furthermore, it was shown that the CMD-s-s-peptide conjugates were highly accumulated in tumor tissue in a mouse xenograft model using BxPC-3 cells, and the anti-tumor activity of the conjugate was more effective than that of the free peptide. In addition, the plasma concentrations of peptide were moderately increased and the elimination half-life of the peptide was prolonged after intravenous injection of CMD-s-s-peptide conjugates. These results demonstrated that the conjugate based on thiolated CMD polymer would be potentially useful carriers for the sustained release of the hybrid peptide in vivo.

P-glycoprotein inhibition by the multidrug resistance-reversing agent MS-209 enhances bioavailability and antitumor efficacy of orally administered paclitaxel.

PURPOSE: Recent studies in humans and mice have demonstrated that intestinal P-glycoprotein plays a causative role in the limited absorption of orally administered paclitaxel. Multidrug resistance (MDR)-reversing agents, such as cyclosporin A and PSC 833, are known to increase the systemic exposure to orally administered paclitaxel by enhancing absorption in the intestinal tract and decreasing elimination via the biliary tract. In this study, we demonstrated that coadministration of the MDR-reversing agent MS-209, which is known to inhibit P-glycoprotein function by direct interaction, improved the bioavailability of orally administered paclitaxel and consequently enhanced its antitumor activity. METHODS: The pharmacokinetics of paclitaxel were examined by measuring [(3)H]paclitaxel in plasma drawn from rats and mice given the drug with or without MS-209. The influence of MS-209 on the intestinal transport of [(3)H]paclitaxel was studied using a human colorectal cancer cell line, Caco-2. The in vivo efficacy of orally administered paclitaxel in combination with MS-209 was further evaluated in B16 melanoma-bearing mice. RESULTS: The plasma concentration of [(3)H]paclitaxel following oral administration was significantly increased by coadministration of MS-209 at 100 mg/kg in both rats and mice. In rats, the AUC of [(3)H]paclitaxel following oral administration was strikingly increased (1.9-fold) by coadministration of MS-209, whereas the AUC of [(3)H]paclitaxel following i.v. injection was slightly increased (1.3-fold) by MS-209. The increase in apparent bioavailability of oral paclitaxel due to MS-209 was 1.4-fold. To demonstrate this enhancing action in vitro, we studied the influence of MS-209 on the transport of [(3)H]paclitaxel using Caco-2 cells, which is a well-known model of intestinal efflux. The transport of [(3)H]paclitaxel across the Caco-2 monolayer was markedly inhibited in the presence of MS-209, and the apparent K(i)of MS-209 for the active transport of [(3)H]paclitaxel was 0.4 microM. Moreover, paclitaxel administered orally at 100 mg/kg per day with MS-209 at 100 mg/kg per day showed significant antitumor activity in B16 melanoma-bearing mice, whereas paclitaxel administered orally alone at the same dose showed no antitumor activity. These results suggest that the coadministration of MS-209 improved low systemic exposure to paclitaxel through inhibition of P-glycoprotein, which is involved in drug excretion via the intestinal tract, resulting in a clear antitumor activity of paclitaxel administered orally. CONCLUSION: The present study suggests that coadministration of MS-209 may be a useful way to improve the bioavailability of drugs not suitable for oral administration due to elimination via the intestinal tract.

The membrane-lytic peptides K8L9 and melittin enter cancer cells via receptor endocytosis following subcytotoxic exposure.

We investigate the cell entry mechanism of the membrane-lytic peptides K8L9 and melittin in cancer cell lines. K8L9 and melittin interacted with the highly expressed endocytic receptors neuropilin-1, low-density lipoprotein-related protein receptor 1 (LRP1), and transferrin receptor. Silencing of these receptors by small interfering RNAs (siRNAs) attenuated the cytotoxic activity of K8L9 in four cancer cell lines. Intracellular K8L9 and melittin triggered enlargement of the lysosomal compartments and cytosolic translocation of cathepsin B. Hsc70 was identified as a melittin-interactive molecule using coimmunoprecipitation and mass spectrometry, and Hsc70-siRNA attenuated the cellular uptake of K8L9 and cytotoxic activity by K8L9 and melittin. These findings suggest that K8L9 and melittin can enter cancer cells via receptor endocytosis following subcytotoxic treatment and subsequently affect lysosomal compartments.

Combination of hybrid peptide with biodegradable gelatin hydrogel for controlled release and enhancement of anti-tumor activity in vivo.

We previously reported that the EGFR2R-lytic hybrid peptide has cytotoxic and anti-tumor activities both in vitro and in vivo. In this study, to improve the peptide pharmacokinetics and its anti-tumor activity after intravenous injection, we prepared biodegradable gelatin hydrogel nanoparticles as the delivery system of peptide. The complex is formed through the electrostatic interaction between the cationic peptide and anionic gelatin. In vitro release studies confirmed that the peptide was released from the complex in phosphate-buffered saline (PBS) solution containing fetal bovine serum at 37°C within 48h, whereas little release was observed in PBS solution. In vivo release studies indicated that the anti-tumor activity of the complex was more effective than that of peptide treatment alone, and high tumor accumulation of the peptide was observed in the mice treated with the complex. Furthermore, the plasma area under the concentration curve (AUC) and half-life (T1/2) values of the complex were higher than those of the peptide treatment alone, respectively. These results demonstrate that the rate of peptide release was controlled by the gelatin, and that the complex had a longer circulation time and enhanced its anti-tumor activity in vivo.