Summary of study ST001731

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001108. The data can be accessed directly via it's Project DOI: 10.21228/M8GM5B This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST001731
Study TitleMitochondrial ATP fuels ABC transporter-mediated drug efflux in cancer chemoresistance (part-II)
Study SummaryChemotherapy remains the standard of care for most cancers worldwide, however development of chemoresistance due to the presence of the drug-effluxing ABC transporters remains a significant problem. The development of safe and effective means to overcome chemoresistance is critical for achieving durable remissions in many cancer patients. We have investigated the energetic demands of ABC transporters in the context of the metabolic adaptations of chemoresistant cancer cells. Here we show that ABC transporters use mitochondrial-derived ATP as a source of energy to efflux drugs out of cancer cells. We further demonstrate that the loss of MCJ (DnaJC15), an endogenous negative regulator of mitochondrial respiration, in chemoresistant cancer cells boosts their ability to produce ATP from mitochondria and fuel ABC transporters. We have developed novel MCJ mimetics that can attenuate mitochondrial respiration and safely overcome chemoresistance in vitro and in vivo. Administration of MCJ mimetics in combination with standard chemotherapeutic drugs could therefore become an new strategy for treatment of multiple cancers.
Institute
University of Colorado Denver
DepartmentBiochemistry and Molecular Genetics
LaboratoryAngelo D'Alessandro
Last NameCulp-Hill
First NameRachel
Address12801 E 17th Ave L18-9403D, Aurora, Colorado, 80045, USA
Emailrachel.hill@cuanschutz.edu
Phone303-724-5798
Submit Date2021-03-18
Raw Data AvailableYes
Raw Data File Type(s).raw
Analysis Type DetailLC-MS
Release Date2021-03-31
Release Version1
Rachel Culp-Hill Rachel Culp-Hill
https://dx.doi.org/10.21228/M8GM5B
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR001108
Project DOI:doi: 10.21228/M8GM5B
Project Title:Mitochondrial ATP fuels ABC transporter-mediated drug efflux in cancer chemoresistance
Project Summary:Chemotherapy remains the standard of care for most cancers worldwide, however development of chemoresistance due to the presence of the drug-effluxing ABC transporters remains a significant problem. The development of safe and effective means to overcome chemoresistance is critical for achieving durable remissions in many cancer patients. We have investigated the energetic demands of ABC transporters in the context of the metabolic adaptations of chemoresistant cancer cells. Here we show that ABC transporters use mitochondrial-derived ATP as a source of energy to efflux drugs out of cancer cells. We further demonstrate that the loss of MCJ (DnaJC15), an endogenous negative regulator of mitochondrial respiration, in chemoresistant cancer cells boosts their ability to produce ATP from mitochondria and fuel ABC transporters. We have developed novel MCJ mimetics that can attenuate mitochondrial respiration and safely overcome chemoresistance in vitro and in vivo. Administration of MCJ mimetics in combination with standard chemotherapeutic drugs could therefore become an new strategy for treatment of multiple cancers.
Institute:University of Colorado Denver
Department:Biochemistry and Molecular Genetics
Laboratory:Angelo D'Alessandro
Last Name:Culp-Hill
First Name:Rachel
Address:12801 E 17th Ave L18-9403D, Aurora, Colorado, 80045, USA
Email:rachel.hill@cuanschutz.edu
Phone:303-724-5798

Subject:

Subject ID:SU001808
Subject Type:Cultured cells
Subject Species:Homo sapiens
Taxonomy ID:9606

Factors:

Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id Factor
SA162629control1Control
SA162630control4Control
SA162631control3Control
SA162632control2Control
SA162633siMCJ4siMCJkd
SA162634siMCJ1siMCJkd
SA162635siMCJ2siMCJkd
SA162636siMCJ3siMCJkd
Showing results 1 to 8 of 8

Collection:

Collection ID:CO001801
Collection Summary:MCF7 cells were purchased from American Type Culture Collection (ATCC). All cancer cell lines were maintained in RPMI 1640 (Sigma R8758) containing glucose (2 mg/ml) and glutamine (0.6 mg/ml) but no pyruvate and was supplemented with 5% Fetal calf serum.
Sample Type:Cultured cells

Treatment:

Treatment ID:TR001821
Treatment Summary:MCF7 cell transfections were performed using siPORT NeoFX Transfection Agent (ThermoFisher Scientific) following the recommended protocol for an oligonucleotide siRNA based on the previously described MCJ targeting sequence.

Sample Preparation:

Sampleprep ID:SP001814
Sampleprep Summary:All cancer cell lines were maintained in RPMI 1640 (Sigma R8758) containing glucose (2 mg/ml) and glutamine (0.6 mg/ml) but no pyruvate and was supplemented with 5% Fetal calf serum. Metabolic profile comparisons of cells were performed on equal cell numbers. Cells were cultured under normal conditions, detached using trypsin-EDTA (0.05 %), counted, normalized to 0.5 x 10e6 in each sample, and cell pellets were snap frozen in liquid nitrogen prior to analysis. Briefly, 2 x 10e6 cells and 20μL cell media were extracted in 1mL and 980μL cold lysis and extraction buffer (methanol : acetonitrile : water, 5:3:2), respectively, and pellets were discarded.

Combined analysis:

Analysis ID AN002817 AN002818
Analysis type MS MS
Chromatography type Reversed phase Reversed phase
Chromatography system Thermo Vanquish Thermo Vanquish
Column Phenomenex Kinetex C18 (150 x 2.1mm, 2.6 um) Phenomenex Kinetex C18 (150 x 2.1mm, 2.6 um)
MS Type ESI ESI
MS instrument type Orbitrap Orbitrap
MS instrument name Thermo Q Exactive Orbitrap Thermo Q Exactive Orbitrap
Ion Mode POSITIVE NEGATIVE
Units Relative Abundance Relative Abundance

Chromatography:

Chromatography ID:CH002082
Chromatography Summary:Positive Mode 10 μL of water and methanol soluble fractions were run through a Kinetex C18 1.7 μm, 100 x 2.1 mm (Phenomenex) reversed phase column (Positive ion mode - phase A: water, 0.1 % formic acid; B: acetonitrile, 0.1 % formic acid) via an ultra-high performance chromatographic system (UHPLC - Vanquish, Thermo Fisher). UHPLC was coupled in line with a high-resolution quadrupole Orbitrap instrument run in positive polarity mode (QExactive, Thermo Fisher) at 70,000 resolution (at 200 m/z). Gradients and other technical parameters and the variants employed herein have been extensively described.
Instrument Name:Thermo Vanquish
Column Name:Phenomenex Kinetex C18 (150 x 2.1mm, 2.6 um)
Chromatography Type:Reversed phase
  
Chromatography ID:CH002083
Chromatography Summary:Negative Mode 10 μL of water and methanol soluble fractions were run through a Kinetex C18 1.7 μm, 100 x 2.1 mm (Phenomenex) reversed phase column (Negative ion mode – phase A: 1 mM NH4Ac 95:5 water : acetonitrile; phase B: 1 mM NH4Ac 95:5 acetonitrile : water) via an ultra-high performance chromatographic system (UHPLC - Vanquish, Thermo Fisher). UHPLC was coupled in line with a high-resolution quadrupole Orbitrap instrument run in negative polarity mode (QExactive, Thermo Fisher) at 70,000 resolution (at 200 m/z). Gradients and other technical parameters and the variants employed herein have been extensively described.
Instrument Name:Thermo Vanquish
Column Name:Phenomenex Kinetex C18 (150 x 2.1mm, 2.6 um)
Chromatography Type:Reversed phase

MS:

MS ID:MS002611
Analysis ID:AN002817
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:UHPLC was coupled in line with a high-resolution quadrupole Orbitrap instrument run in positive polarity mode (QExactive, Thermo Fisher) at 70,000 resolution (at 200 m/z). Gradients and other technical parameters and the variants employed herein have been extensively described.
Ion Mode:POSITIVE
  
MS ID:MS002612
Analysis ID:AN002818
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:UHPLC was coupled in line with a high-resolution quadrupole Orbitrap instrument run in negative polarity mode (QExactive, Thermo Fisher) at 70,000 resolution (at 200 m/z). Gradients and other technical parameters and the variants employed herein have been extensively described.
Ion Mode:NEGATIVE
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