In vivo imaging of multidrug resistance using a third generation MDR1 inhibitor.
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ABSTRACT:
SUBMITTER: Sprachman MM
PROVIDER: S-EPMC4098115 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The human multidrug-resistance (MDR1) P-glycoprotein (Pgp) is an ATP-binding-cassette transporter (ABCB1) that is ubiquitously expressed. Often its concentration is high in the plasma membrane of cancer cells, where it causes multidrug resistance by pumping lipophilic drugs out of the cell. In addition, MDR1 Pgp can transport analogues of membrane lipids with shortened acyl chains across the plasma membrane. We studied a role for MDR1 Pgp in transport to the cell surface of the signal-transduction molecule platelet-activating factor (PAF). PAF is the natural short-chain phospholipid 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine. [(14)C]PAF synthesized intracellularly from exogenous alkylacetylglycerol and [(14)C]choline became accessible to albumin in the extracellular medium of pig kidney epithelial LLC-PK1 cells in the absence of vesicular transport. Its translocation across the apical membrane was greatly stimulated by the expression of MDR1 Pgp, and inhibited by the MDR1 inhibitors PSC833 and cyclosporin A. Basolateral translocation was not stimulated by expression of the basolateral drug transporter MRP1 (ABCC1). It was insensitive to the MRP1 inhibitor indomethacin and to depletion of GSH which is required for MRP1 activity. While efficient transport of PAF across the apical plasma membrane may be physiologically relevant in MDR1-expressing epithelia, PAF secretion in multidrug-resistant tumours may stimulate angiogenesis and thereby tumour growth.
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Project description:Resistance of Lactococcus lactis to cytotoxic compounds shares features with the multidrug resistance phenotype of mammalian tumor cells. Here, we report the gene cloning and functional characterization in Escherichia coli of LmrA, a lactococcal structural and functional homolog of the human multidrug resistance P-glycoprotein MDR1. LmrA is a 590-aa polypeptide that has a putative topology of six alpha-helical transmembrane segments in the N-terminal hydrophobic domain, followed by a hydrophilic domain containing the ATP-binding site. LmrA is similar to each of the two halves of MDR1 and may function as a homodimer. The sequence conservation between LmrA and MDR1 includes particular regions in the transmembrane domains and connecting loops, which, in MDR1 and the MDR1 homologs in other mammalian species, have been implicated as determinants of drug recognition and binding. LmrA and MDR1 extrude a similar spectrum of amphiphilic cationic compounds, and the activity of both systems is reversed by reserpine and verapamil. As LmrA can be functionally expressed in E. coli, it offers a useful prokaryotic model for future studies on the molecular mechanism of MDR1-like multidrug transporters.
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Project description:Multiphoton microscopy using laser sources in the mid-infrared range (MIR, 1,300 nm and 1,700 nm) was used to image atherosclerotic plaques from murine and human samples. Third harmonic generation (THG) from atherosclerotic plaques revealed morphological details of cellular and extracellular lipid deposits. Simultaneous nonlinear optical signals from the same laser source, including second harmonic generation and endogenous fluorescence, resulted in label-free images of various layers within the diseased vessel wall. The THG signal adds an endogenous contrast mechanism with a practical degree of specificity for atherosclerotic plaques that complements current nonlinear optical methods for the investigation of cardiovascular disease. Our use of whole-mount tissue and backward scattered epi-detection suggests THG could potentially be used in the future as a clinical tool.
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Project description:Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are effective in patients with non-small-cell lung cancer (NSCLC) harboring EGFR mutations. However, due to acquired resistance to EGFR-TKIs, even patients on third-generation osimertinib have a poor prognosis. Resistance mechanisms are still not fully understood. Here, we demonstrate that the increased expression of MUSASHI-2 (MSI2), an RNA-binding protein, is a novel mechanism for resistance to EGFR-TKIs. We found that after a long-term exposure to gefitinib, the first-generation EGFR-TKI lung cancer cells harboring the EGFR-TKI-sensitive mutations became resistant to both gefitinib and osimertinib. Although other mutations in EGFR were not found, expression levels of Nanog, a stemness core protein, and activities of aldehyde dehydrogenase (ALDH) were increased, suggesting that cancer stem-like properties were increased. Transcriptome analysis revealed that MSI2 was among the stemness-related genes highly upregulated in EGFR-TKI-resistant cells. Knockdown of MSI2 reduced cancer stem-like properties, including the expression levels of Nanog, a core stemness factor. We demonstrated that knockdown of MSI2 restored sensitivity to osimertinib or gefitinib in EGFR-TKI-resistant cells to levels similar to those of parental cells in vitro. An RNA immunoprecipitation (RIP) assay revealed that antibodies against MSI2 were bound to Nanog mRNA, suggesting that MSI2 increases Nanog expression by binding to Nanog mRNA. Moreover, overexpression of MSI2 or Nanog conferred resistance to osimertinib or gefitinib in parental cells. Finally, MSI2 knockdown greatly increased the sensitivity to osimertinib in vivo. Collectively, our findings provide proof of principle that targeting the MSI2-Nanog axis in combination with EGFR-TKIs would effectively prevent the emergence of acquired resistance.
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Project description:PurposeLorlatinib is a third-generation anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor with proven efficacy in patients with ALK-rearranged lung cancer previously treated with first- and second-generation ALK inhibitors. Beside compound mutations in the ALK kinase domain, other resistance mechanisms driving lorlatinib resistance remain unknown. We aimed to characterize the mechanisms of resistance to lorlatinib occurring in patients with ALK-rearranged lung cancer and design new therapeutic strategies in this setting.Experimental designResistance mechanisms were investigated in 5 patients resistant to lorlatinib. Longitudinal tumor biopsies were studied using high-throughput next-generation sequencing. Patient-derived models were developed to characterize the acquired resistance mechanisms, and Ba/F3 cell mutants were generated to study the effect of novel ALK compound mutations. Drug combinatory strategies were evaluated in vitro and in vivo to overcome lorlatinib resistance.ResultsDiverse biological mechanisms leading to lorlatinib resistance were identified. Epithelial-mesenchymal transition (EMT) mediated resistance in two patient-derived cell lines and was susceptible to dual SRC and ALK inhibition. We characterized three ALK kinase domain compound mutations occurring in patients, L1196M/D1203N, F1174L/G1202R, and C1156Y/G1269A, with differential susceptibility to ALK inhibition by lorlatinib. We identified a novel bypass mechanism of resistance caused by NF2 loss-of-function mutations, conferring sensitivity to treatment with mTOR inhibitors.ConclusionsThis study shows that mechanisms of resistance to lorlatinib are diverse and complex, requiring new therapeutic strategies to tailor treatment upon disease progression.
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