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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/1471-2407-14-615) contains supplementary material, which is available to authorized users

Generation of Tetrafluoroethylene–Propylene Elastomer-Based Microfluidic Devices for Drug Toxicity and Metabolism Studies

フッ素系エラストマー素材を用いた肝臓チップの開発と薬物代謝・毒性試験への応用. 京都大学プレスリリース. 2021-09-16.Drug testing on miniatured livers. 京都大学プレスリリース. 2021-09-17.Polydimethylsiloxane (PDMS) is widely used to fabricate microfluidic organs-on-chips. Using these devices (PDMS-based devices), the mechanical microenvironment of living tissues, such as pulmonary respiration and intestinal peristalsis, can be reproduced in vitro. However, the use of PDMS-based devices in drug discovery research is limited because of their extensive absorption of drugs. In this study, we investigated the feasibility of the tetrafluoroethylene–propylene (FEPM) elastomer to fabricate a hepatocyte-on-a-chip (FEPM-based hepatocyte chip) with lower drug absorption. The FEPM-based hepatocyte chip expressed drug-metabolizing enzymes, drug-conjugating enzymes, and drug transporters. Also, it could produce human albumin. Although the metabolites of midazolam and bufuralol were hardly detected in the PDMS-based hepatocyte chip, they were detected abundantly in the FEPM-based hepatocyte chip. Finally, coumarin-induced hepatocyte cytotoxicity was less severe in the PDMS-based hepatocyte chip than in the FEPM-based hepatocyte chip, reflecting the different drug absorptions of the two chips. In conclusion, the FEPM-based hepatocyte chip could be a useful tool in drug discovery research, including drug metabolism and toxicity studies

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

Abstract 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.</p

Monitoring of Bip promoter activation during cancer cell growth by bioluminescence imaging technique at single cell level

Cancer cells require the regulation of organelle-specific unfolded protein responses, such as endoplasmic reticulum (ER) stress, because of their increased metabolic activity during rapid proliferation and cell growth, which are executed through the activation of diverse signaling pathways. In this study, we focused on the dynamic regulation of ER stress in accordance with cancer cellular demand, and we performed real-time monitoring of the activation of the binding immunoglobulin protein (Bip) promoter, which is one of the most responsive genes to ER stress during cancer cell growth, in two and three dimensional (2D and 3D) cell culture using bioluminescence imaging at the single-cell level. Bioluminescence images were obtained from living single cancer cells after transient transfection of the reporter gene, and we observed Bip promoter activation during cell growth. Bip promoter activation was also observed in 2D and 3D culture using stably transfected glioblastoma cancer cells with the reporter gene. The Bip promoter was activated especially in dividing cells during cell growth. We then performed real-time monitoring of Bip promoter activation by bioluminescence imaging in tissue slices obtained from U251/pBipPro-Luc tumors. Luminescence intensity was not constant and was different in individual regions of the tumor slices, and the Bip promoter was activated in several regions during monitoring in vitro. These results show that real-time monitoring by bioluminescence imaging at the single-cell level is a suitable tool for not only gene analysis of signal transduction and regulation of the dynamics of the unfolded protein response in cancer cells but also for the evaluation of the efficacy of anti-cancer agents, and could provide additional information that has been difficult to obtain using conventional assays

Tetrafluoroethylene-Propylene Elastomer for Fabrication of Microfluidic Organs-on-Chips Resistant to Drug Absorption

Organs-on-chips are microfluidic devices typically fabricated from polydimethylsiloxane (PDMS). Since PDMS has many attractive properties including high optical clarity and compliance, PDMS is very useful for cell culture applications; however, PDMS possesses a significant drawback in that small hydrophobic molecules are strongly absorbed. This drawback hinders widespread use of PDMS-based devices for drug discovery and development. Here, we describe a microfluidic cell culture system made of a tetrafluoroethylene-propylene (FEPM) elastomer. We demonstrated that FEPM does not absorb small hydrophobic compounds including rhodamine B and three types of drugs, nifedipine, coumarin, and Bay K8644, whereas PDMS absorbs them strongly. The device consists of two FEPM layers of microchannels separated by a thin collagen vitrigel membrane. Since FEPM is flexible and biocompatible, this microfluidic device can be used to culture cells while applying mechanical strain. When human umbilical vein endothelial cells (HUVECs) were subjected to cyclic strain (~10%) for 4 h in this device, HUVECs reoriented and aligned perpendicularly in response to the cyclic stretch. Moreover, we demonstrated that this device can be used to replicate the epithelial&ndash;endothelial interface as well as to provide physiological mechanical strain and fluid flow. This method offers a robust platform to produce organs-on-chips for drug discovery and development

Tetrafluoroethylene-Propylene Elastomer for Fabrication of Microfluidic Organs-on-Chips Resistant to Drug Absorption

Organs-on-chips are microfluidic devices typically fabricated from polydimethylsiloxane (PDMS). Since PDMS has many attractive properties including high optical clarity and compliance, PDMS is very useful for cell culture applications; however, PDMS possesses a significant drawback in that small hydrophobic molecules are strongly absorbed. This drawback hinders widespread use of PDMS-based devices for drug discovery and development. Here, we describe a microfluidic cell culture system made of a tetrafluoroethylene-propylene (FEPM) elastomer. We demonstrated that FEPM does not absorb small hydrophobic compounds including rhodamine B and three types of drugs, nifedipine, coumarin, and Bay K8644, whereas PDMS absorbs them strongly. The device consists of two FEPM layers of microchannels separated by a thin collagen vitrigel membrane. Since FEPM is flexible and biocompatible, this microfluidic device can be used to culture cells while applying mechanical strain. When human umbilical vein endothelial cells (HUVECs) were subjected to cyclic strain (~10%) for 4 h in this device, HUVECs reoriented and aligned perpendicularly in response to the cyclic stretch. Moreover, we demonstrated that this device can be used to replicate the epithelial–endothelial interface as well as to provide physiological mechanical strain and fluid flow. This method offers a robust platform to produce organs-on-chips for drug discovery and development.</jats:p