title: The inhibitory and combinative mechanism of HZ08 with P-glycoprotein expressed on the membrane of Caco-2 cell line.
abstract: Recently, the research and development of agents to reverse the phenomenon of multidrug resistance has been an attractive goal as well as a key approach to elevating the clinical survival of cancer patients. Although three generations of P-glycoprotein modulators have been identified, poor clearance and metabolism render these agents too toxic to be used in clinical application. HZ08, which has been under investigation for several years, shows a dramatic reversal effect with low cytotoxicity. For the first time, we aimed to describe the interaction between HZ08 and P-glycoprotein in Caco-2 cell line in which P-glycoprotein is overexpressed naturally. Cytotoxicity and multidrug resistance reversal assays, together with flow cytometry, fluorescence microscopy and siRNA interference as well as Caco-2 monolayer transport model were employed in this study to evaluate the interaction between HZ08 and P-glycoprotein. This study revealed that HZ08 was capable of reversing adriamycin resistance mediated by P-glycoprotein as a result of intracellular enhancement of adriamycin accumulation, which was found to be superior to verapamil. In addition, we confirmed that HZ08 suppressed the transport of Rhodamine123 in the Caco-2 monolayer model but had little effect on P-glycoprotein expression. The transport of HZ08 was diminished by P-glycoprotein inhibitors (verapamil and LY335979) and its accumulation was increased via siRNA targeting MDR1 in Caco-2 cells. Furthermore, considering the binding site of P-glycoprotein, verapamil performed as a competitive inhibitor with HZ08. In conclusion, as a P-glycoprotein substrate, HZ08 inhibited P-glycoprotein activity and may share the same binding site of verapamil to P-glycoprotein.
authors: State Key Laboratory of Natural Medicines, Department of Physiology, China Pharmaceutical University, Nanjing 210009, PR China.
authors: Yanyan
authors: Y
authors: Zhang
authors: State Key Laboratory of Natural Medicines, Department of Physiology, China Pharmaceutical University, Nanjing 210009, PR China.
authors: Yahui
authors: Y
authors: Hu
authors: State Key Laboratory of Natural Medicines, Department of Physiology, China Pharmaceutical University, Nanjing 210009, PR China.
authors: Yidong
authors: Y
authors: Feng
authors: State Key Laboratory of Natural Medicines, Department of Physiology, China Pharmaceutical University, Nanjing 210009, PR China.
authors: Nandani Darshika
authors: ND
authors: Kodithuwakku
authors: State Key Laboratory of Natural Medicines, Department of Physiology, China Pharmaceutical University, Nanjing 210009, PR China.
authors: Weirong
authors: W
authors: Fang
authors: State Key Laboratory of Natural Medicines, Department of Physiology, China Pharmaceutical University, Nanjing 210009, PR China. Electronic address: yunmanlicpu@hotmail.com.
authors: Yunman
authors: Y
authors: Li
authors: Center of Drug Discovery, China Pharmaceutical University, Nanjing 210009, PR China.
authors: Wenlong
authors: W
authors: Huang
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D001665:Binding Sites; D001692:Biological Transport; D018938:Caco-2 Cells; D002462:Cell Membrane / Q000187:drug effects* / Q000378:metabolism; D002853:Chromatography, Liquid; D003986:Dibenzocycloheptenes / Q000494:pharmacology; D004317:Doxorubicin / Q000494:pharmacology; D018432:Drug Resistance, Multiple; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology*; D013058:Mass Spectrometry; D008856:Microscopy, Fluorescence; D011804:Quinolines / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000235:genetics; D020112:Rhodamine 123 / Q000493:pharmacokinetics; D014700:Verapamil / Q000494:pharmacology
chemical_list: C513055:ABCB1 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000903:Antibiotics, Antineoplastic; D003986:Dibenzocycloheptenes; D007546:Isoquinolines; C524509:N-cyano-1-((3,4-dimethoxyphenyl)methyl)-3,4-dihydro-6,7-dimethoxy-N'-octyl-2(1H)-isoquinoline-carboximidamide; D011804:Quinolines; D034741:RNA, Small Interfering; D020112:Rhodamine 123; D004317:Doxorubicin; C095179:zosuquidar trihydrochloride; D014700:Verapamil
keywords: Competitive substrate; HZ08; Inhibitor; Multidrug resistance; P-glycoprotein