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BackgroundDespite 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.MethodsWe 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.ResultsIt 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.ConclusionThese results indicate that Antp-TPR peptide would provide a potent and selective anticancer therapy to cancer patients.

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Immunoglobulin E (IgE) bound to multivalent antigen (Ag) elicits mast cell degranulation but not survival; on the contrary, IgE in the absence of Ag (IgE(-Ag)) induces survival only but not degranulation. Although these distinct responses are mediated through the same receptor, Fc⑀RI, the molecular mechanism generating the divergence is largely unknown. We recently showed that the signals through FcR␥ chain are essential for IgE(-Ag)-induced mast cell survival as well as IgE(؉Ag)-induced degranulation.To determine whether the cellular output is regulated by the quantity of FcR␥ signal, we expressed CD8/FcR␥ chimeras (CD8/␥) in bone marrow-derived mast cells (BMMCs) from FcR␥ ؊/؊ mice to manipulate the strength of FcR␥ signals by anti-CD8 cross-linking. Cross-linking of CD8/␥ induced mast cell survival and degranulation. Survival was induced by weaker stimulation than needed for degranulation in terms of anti-CD8 concentration and the valency of chimera. However, sustained extracellular signalregulated kinase (Erk) activation seems to regulate survival even when the activation signal was strong enough to elicit degranulation. Generation of sustained Erk activation by active mitogen-activated protein kinase kinase (MEK) induced BMMC survival. These results suggest that the duration and the magnitude of FcR␥ signals may determine mast cell survival and degranulation, respectively. IntroductionImmunoglobulin E (IgE) triggers antiparasitic immunity or allergic responses via binding to Fc⑀RI on the surface of mast cells and basophils. 1 Rodent Fc⑀RI is expressed as a tetramer composed of specific ␣, ␤ (Fc⑀RI␣, Fc⑀RI␤), and common ␥ (FcR␥) homodimers shared with Fc␥RI or Fc␥RIII. 2 The ␣ chain mediates binding to IgE. The ␤ and ␥ chains possess immunoreceptor tyrosine-based activation motifs (ITAMs) 3 within their cytoplasmic domains. Cross-linking of Fc⑀RI with IgE and multivalent antigen (IgE(ϩAg)) initiates an activation signal cascade via tyrosine phosphorylation of these ITAMs by Lyn. Syk is then recruited to the phospho-ITAMs of Fc⑀RI␥, where it is activated to phosphorylate various substrates in the downstream cascade, which leads to degranulation or cytokine production. 1,4 It is recently reported that IgE in the absence of Ag (IgE(-Ag)) actively promotes mast cell survival in addition to passive sensitization. 5,6 IgE(ϩAg) can evoke degranulation but not survival, whereas IgE(-Ag) induces survival but not degranulation. 6 Although both these responses are mediated through Fc⑀RI, the molecular mechanism underlying it remains to be fully elucidated. 6,7 We have recently demonstrated that ITAM in the FcR␥ chain is essential for IgE(-Ag)-induced mast cell survival as well as degranulation and cytokine production evoked by IgE(ϩAg). 50 This suggests that distinct cellular responses like mediator release and survival are triggered by a similar signaling mechanism, in an FcR␥ ITAM-dependent manner. One possible hypothesis for the distinct responses mediated by the same receptor is that the strength of FcR␥ signals d...

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BackgroundTransferrin 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.MethodsIn 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.ResultsThe TfR-lytic hybrid peptide showed cytotoxic activity in 12 cancer cell lines, with IC50 values as low as 4.0-9.3 μM. Normal cells were less sensitive to this molecule, with IC50 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.ConclusionsTfR-lytic peptide might provide a potent and selective anticancer therapy for patients.

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.

BackgroundHeat-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.ResultsIn 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.ConclusionThese 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.