title: Multi-stage inhibition in breast cancer metastasis by orally active triple conjugate, LHTD4 (low molecular weight heparin-taurocholate-tetrameric deoxycholate).
abstract: Targeting multiple stages in metastatic breast cancer is one of the effective ways to inhibit metastatic progression. To target human metastatic breast cancer as well as improving patient compliance, we developed an orally active low molecular weight heparin (LMWH)-taurocholate conjugated with tetrameric deoxycholic acid, namely LHTD4, which followed by physical complexation with a synthetic bile acid enhancer, DCK. In breast cancer, both transforming growth factor-β1 (TGF-β1) and CXCL12 exhibit enhanced metastatic activity during the initiation and progression stages of breast cancer, thus we direct the focus on investigating the antimetastatic effect of LHTD4/DCK complex by targeting TGF-β1 and CXCL12. Computer simulation study and SPR analysis were performed for the binding confirmation of LHTD4 with TGF-β1 and CXCL12. We carried out in vitro phosphorylation assays of the consecutive receptors of TGF-β1 and CXCL12 (TGF-β1R1 and CXCR4, respectively). Effects of LHTD4 on in vitro cell migration (induced by TGF-β1) and chemotaxis (mediated by CXCL12) were investigated. The in vivo anti-metastatic effect of LHTD4 was evaluated in an accelerated metastasis model and an orthotopic MDA-MB-231 breast cancer model. The obtained KD values of TGF-β1 and CXCL12 with LHTD4 were 0.85 and 0.019 μM respectively. The simulation study showed that binding affinities of LHTD4 fragment with either TGF-β1 or CXCL12 through additional electrostatic interaction was more stable than that of LMWH fragment. In vitro phosphorylation assays of TGF-β1R1 and CXCR4 showed that the effective inhibition of receptor phosphorylation was observed with the treatment of LHTD4. The expressions of epithelial to mesenchymal transition (EMT) marker proteins such as vimentin and Snail were prevented by LTHD4 treatment in in vitro studies with TGF-β1 treated MDA-MB-231 cells. Moreover, we observed that LHTD4 negatively regulated the functions of TGF-β1 and CXCL12 on migration and invasion of breast cancer cell. In several advanced orthotopic and experimental breast cancer metastasis murine models, the treatment with LHTD4 (5 mg/kg daily, p.o.) significantly inhibited metastasis compared to the control. Overall, LHTD4 exhibited anti-metastatic effects by inhibiting TGF-β1 and CXCL12, and the clinically relevant dose of orally active LHTD4 was found to be effective in preclinical studies without any apparent toxicity.
authors.affiliation: Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, College of Pharmacy, Seoul National University, Seoul, South Korea.
authors.forename: Farzana
authors.initials: F
authors.lastname: Alam
authors.affiliation: Biomedical Research Institute, Korea Institute of Science and Technology (KIST), Seoul, South Korea.
authors.forename: Taslim A
authors.initials: TA
authors.lastname: Al-Hilal
authors.affiliation: Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul, South Korea.
authors.forename: Jooho
authors.initials: J
authors.lastname: Park
authors.affiliation: Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul, South Korea.
authors.forename: Jeong Uk
authors.initials: JU
authors.lastname: Choi
authors.affiliation: Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, College of Pharmacy, Seoul National University, Seoul, South Korea.
authors.forename: Foyez
authors.initials: F
authors.lastname: Mahmud
authors.affiliation: College of Pharmacy, Yeungnam University, Gyeongsan, South Korea.
authors.forename: Jee-Heon
authors.initials: JH
authors.lastname: Jeong
authors.affiliation: Biomedical Research Institute, Korea Institute of Science and Technology (KIST), Seoul, South Korea.
authors.forename: In-San
authors.initials: IS
authors.lastname: Kim
authors.affiliation: Biomedical Research Institute, Korea Institute of Science and Technology (KIST), Seoul, South Korea; Asan Medical Center, Department of Otolaryngology, College of Medicine, University of Ulsan, Seoul, South Korea.
authors.forename: Sang Yoon
authors.initials: SY
authors.lastname: Kim
authors.affiliation: College of Pharmacy, Chosun University, Gwangju, South Korea. Electronic address: srhwang@chosun.ac.kr.
authors.forename: Seung Rim
authors.initials: SR
authors.lastname: Hwang
authors.affiliation: Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, College of Pharmacy, Seoul National University, Seoul, South Korea; Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul, South Korea. Electronic address: yrbyun@snu.ac.kr.
authors.forename: Youngro
authors.initials: Y
authors.lastname: Byun
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology / Q000627:therapeutic use*; D001940:Breast / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D054377:Chemokine CXCL12 / Q000378:metabolism; D003840:Deoxycholic Acid / Q000031:analogs & derivatives / Q000494:pharmacology / Q000627:therapeutic use*; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects; D005260:Female; D006495:Heparin, Low-Molecular-Weight / Q000031:analogs & derivatives / Q000494:pharmacology / Q000627:therapeutic use*; D006801:Humans; D016513:Mice, SCID; D058990:Molecular Targeted Therapy; D009362:Neoplasm Metastasis / Q000473:pathology / Q000517:prevention & control*; D010766:Phosphorylation / Q000187:drug effects; D013656:Taurocholic Acid / Q000031:analogs & derivatives / Q000494:pharmacology / Q000627:therapeutic use*; D053773:Transforming Growth Factor beta1 / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D054377:Chemokine CXCL12; D006495:Heparin, Low-Molecular-Weight; D053773:Transforming Growth Factor beta1; D003840:Deoxycholic Acid; D013656:Taurocholic Acid
keywords: CXCL12; Low molecular weight heparin; Metastasis; Multi-stage targeting; Polymeric bile acid; TGF-β1