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1.
J Cancer Res Clin Oncol ; 146(4): 843-858, 2020 04.
Article in English | MEDLINE | ID: mdl-32056006

ABSTRACT

PURPOSE: Increased ATP-binding-cassette (ABC) transporter activity is a major cause of chemotherapy resistance in cancer. The ABC transporter family member ABCB1 is often overexpressed in colorectal cancer (CRC). Phosphatidylinositol-4,5-bisphosphat (PI(4,5)P2)-dependent pathways are involved in the regulation of ABCB1 function. The protein Myristoylated Alanine-Rich C-Kinase Substrate (MARCKS) is a pivotal regulator of PI(4,5)P2 and inactivated in many CRC cancers via genetic deletion or hyperphosphorylation. Therefore, MARCKS may critically impact ABCB1. METHODS: CRC samples as well as CRC cell lines were tested for a connection between MARCKS and ABCB1 via immunofluorescence and Western-blot analysis. ABCB1 function was studied via calcein influx assay under treatment with known ABCB1 inhibitors (verapamil, tariquidar) as well as the kinase inhibitor bosutinib. ABCB1 internalization and MARCKS translocation was analyzed via confocal microscopy exploiting the endocytosis inhibitors chlorpromazine and dynasore. Abundance of PI(4,5)P2 was monitored by intramolecular fluorescence resonance energy transfer (FRET). Reproductive cell survival was studied via colorimetric WST-1 and clonogenic assays in combination with exposure to the chemotherapeutics doxorubicin and 5-fuorouracil (5-FU). RESULTS: We found increased ABCB1 expression in MARCKS negative CRC patient tumor samples and established CRC cell lines. Mechanistically, the reconstitution of MARCKS function via recombinant expression or the pharmacological inhibition of MARCKS phosphorylation led to a substantial decrease in ABCB1 activity. In CRC cells, bosutinib treatment resulted in a MARCKS translocation from the cytosol to the plasma membrane, while simultaneously, ABCB1 was relocated to intracellular compartments. Inhibition of MARCKS phosphorylation via bosutinib rendered cells more sensitive to the chemotherapeutics doxorubicin and 5-FU. CONCLUSIONS: Cells devoid of MARCKS function showed incomplete ABCB1 internalization, leading to higher ABCB1 activity enhancing chemoresistance. Vice versa our data suggest the prevention of MARCKS inhibition by reversing hyperphosphorylation or genomic restoration after deletion as two promising approaches to overcome tumor cell resistance towards chemotherapeutic ABCB1 substrates.


Subject(s)
Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/metabolism , Myristoylated Alanine-Rich C Kinase Substrate/metabolism , ATP Binding Cassette Transporter, Subfamily B/metabolism , Aniline Compounds , Cell Line, Tumor , Drug Resistance, Neoplasm , Fluoresceins/metabolism , Fluorescence Resonance Energy Transfer , HT29 Cells , Humans , Microscopy, Confocal , Myristoylated Alanine-Rich C Kinase Substrate/deficiency , Nitriles , Phosphorylation , Quinolines
2.
Int J Mol Med ; 42(1): 193-207, 2018 Jul.
Article in English | MEDLINE | ID: mdl-29620148

ABSTRACT

Non-alcoholic fatty liver disease (NAFLD) comprises a spectrum of liver damage characterized by abnormal hepatic fat accumulation and inflammatory response. Although the molecular mechanisms responsible for the disease are not yet fully understood, the pathogenesis of NAFLD likely involves multiple signals. The identification of effective therapeutic strategies to target these signals is of utmost importance. Carnosic acid (CA), as a phenolic diterpene with anticancer, anti-bacterial, anti-diabetic and neuroprotective properties, is produced by many species of the Lamiaceae family. Myristoylated alanine-rich C-kinase substrate (MARCKS) is a major protein kinase C (PKC) substrate in many different cell types. In the present study, wild-type C57BL/6 and MARCKS-deficient mice were randomly divided into the normal chow- or high-fat (HF) diet-fed groups. The HF diet increased the fasting glucose and insulin levels, and promoted glucose intolerance in the wild-type mice. MARCKS deficiency further upregulated intolerance, fasting glucose and insulin. The HF diet also promoted hepatic steatosis, serum alanine transaminase (ALT) and aspartate transaminase (AST) activity, inflammation and lipid accumulation in the wild-type mice. These responses were accelerated in the MARCKS-deficient mice. Importantly, increased inflammation and lipid accumulation were associated with phosphoinositide 3-kinase (PI3K)/AKT, NLR family pyrin domain containing 3 (NLRP3)/nuclear factor-κB (NF-κB) and sterol regulatory element binding protein-1c (SREBP-1c) signaling pathway activation. The mice treated with CA exhibited a significantly improved glucose and insulin tolerance. The production of pro-inflammatory cytokines and lipid accumulation were suppressed by CA. Significantly, MARCKS was reduced in mice fed the HF diet. CA treatment upregulated MARCKS expression compared to the HF group. Furthermore, the activation of the PI3K/AKT, NLRP3/NF-κB and SREBP-1c signaling pathways was inhibited by CA. Taken together, our data suggest that CA suppresses inflammation and lipogenesis in mice fed a HF diet through MARCKS regulation. Thus, CA may be prove to be a useful anti-NAFLD agent.


Subject(s)
Abietanes/therapeutic use , Myristoylated Alanine-Rich C Kinase Substrate/metabolism , Non-alcoholic Fatty Liver Disease/drug therapy , Non-alcoholic Fatty Liver Disease/metabolism , Protective Agents/therapeutic use , Abietanes/pharmacology , Animals , Cytokines/metabolism , Diet, High-Fat , Feeding Behavior , Glucose/metabolism , Homeostasis/drug effects , Inflammasomes/metabolism , Inflammation Mediators/metabolism , Insulin Resistance , Lipogenesis/drug effects , Liver/drug effects , Liver/injuries , Male , Mice, Inbred C57BL , Myristoylated Alanine-Rich C Kinase Substrate/deficiency , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Non-alcoholic Fatty Liver Disease/pathology , Phosphatidylinositol 3-Kinases/metabolism , Protective Agents/pharmacology , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction/drug effects
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