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Heterogeneous Off-Target Effects of Ultra-Low Dose Dimethyl Sulfoxide (DMSO) on Targetable Signaling Events in Lung
Elisa Baldelli1, Mahalakshmi Subramanian2, Abduljalil M Alsubaie2
1Center for Applied Proteomics and Molecular Medicine, George Mason University, Manassas, VA 20110, USA.
Abstract:
Targetable alterations in cancer offer novel opportunities to the drug discovery process. However, pre-clinical testing often requires solubilization of these drugs in cosolvents like dimethyl sulfoxide (DMSO). Using a panel of cell lines commonly used for in vitro drug screening and pre-clinical testing, we explored the DMSO off-target effects on functional signaling networks, drug targets, and downstream substrates. Eight Non-Small Cell Lung Cancer (NSCLC) cell lines were incubated with three concentrations of DMSO (0.0008%, 0.002%, and 0.004% ) over time. Expression and activation levels of 187 proteins, of which 137 were kinases and downstream substrates, were captured using the Reverse Phase Protein Array (RPPA). The DMSO effect was heterogeneous across cell lines and varied based on concentration, exposure time, and cell line. Of the 187 proteins measured, all were statistically different in at least one comparison at the highest DMSO concentration, followed by 99.5% and 98.9% at lower concentrations. Only 46% of the proteins were found to be statistically different in more than 5 cell lines, indicating heterogeneous response across models. These cell line specific alterations modulate response to in vitro drug screening. Ultra-low DMSO concentrations have broad and heterogeneous effects on targetable signaling proteins. Off-target effects need to be carefully evaluated in pre-clinical drug screening and testing.
Insights
Dimethyl sulfoxide (DMSO) used in cancer drug screening has unintended effects on cell signaling proteins. These off-target impacts vary by cell line and concentration, potentially altering drug screening results.
Area of Science:
- Oncology
- Pharmacology
- Molecular Biology
Background:
- Targetable cancer alterations present new drug discovery avenues.
- Pre-clinical drug testing often involves solubilizing compounds in dimethyl sulfoxide (DMSO).
Purpose of the Study:
- To investigate the off-target effects of DMSO on signaling networks in cancer cell lines.
- To assess DMSO's impact on drug targets and downstream substrates during in vitro drug screening.
Main Methods:
- Eight Non-Small Cell Lung Cancer (NSCLC) cell lines were treated with varying DMSO concentrations (0.0008% to 0.004%).
- Reverse Phase Protein Array (RPPA) was used to measure expression and activation levels of 187 proteins, including kinases and substrates.
Main Results:
- DMSO exhibited heterogeneous effects across cell lines, varying with concentration and exposure time.
- At the highest concentration, all 187 measured proteins showed statistically significant differences.
- Only 46% of proteins were affected in more than 5 cell lines, highlighting cell line-specific responses.
Conclusions:
- Ultra-low DMSO concentrations induce broad and heterogeneous effects on targetable signaling proteins.
- Cell line-specific alterations caused by DMSO can modulate in vitro drug screening outcomes.
- Careful evaluation of DMSO off-target effects is crucial for accurate pre-clinical drug screening and testing.
