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. 2024 Mar 5;19(3):e0297661.
doi: 10.1371/journal.pone.0297661. eCollection 2024.

ABCG2 transporter reduces protein aggregation in cigarette smoke condensate-exposed A549 lung cancer cells

Emmanuella O Ajenu  1 , Ashley M Seideneck  1 , Esh Pandellapalli  1 , Emily M Shinsky  1 , Casey L Humphries  1 , Nicholas L Aparicio  1 , Mahak Sharma  1 , James H Marden  2 , Maria M Krasilnikova  1
Affiliations

ABCG2 transporter reduces protein aggregation in cigarette smoke condensate-exposed A549 lung cancer cells

Emmanuella O Ajenu et al. PLoS One. .

Abstract

Cigarette smoke-induced protein aggregation damages the lung cells in emphysema and COPD; however, lung cancer cells continue to thrive, evolving to persist in the toxic environment. Here, we showed that upon the cigarette smoke condensate exposure, A549 lung cancer cells exhibit better survival and reduced level of protein aggregation when compared to non-cancerous Beas-2B and H-6053 cells. Our data suggests that upregulation of efflux pumps in cancer cells assists in reducing smoke toxicity. Specifically, we demonstrated that inhibition of the ABCG2 transporter in A549 by febuxostat or its downregulation by shRNA-mediated RNA interference resulted in a significant increase in protein aggregation due to smoke exposure.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Protein aggregation caused by CSC exposure is less in cancer cells A549, than normal monkey kidney CV1 cells.
The cells were grown in the presence of 0.3 μl of CSC for 24 hours, then stained with Proteostat Aggresome Detection kit.
Fig 2
Fig 2. Comparison of protein aggregation in lung cancer cell line A549 and non-cancer cell lines Beas-2B and H-6053 across different CSC concentrations.
The cells were exposed to different concentrations of CSC for 24 hours. A. Protein aggregates, stained by Proteostat Aggresome Detection kit. B. NFI of the protein aggregates shown in A.
Fig 3
Fig 3. Growth curves of A549, Beas2B, and H-6053 in different concentrations of smoke.
The time dependence of the average cell numbers in 15.2 mm2 area corresponding to the field of view of the microscope screen with 4x objective.
Fig 4
Fig 4. Time dependence of protein aggregation in lung cell lines.
A. Protein aggregation in Beas-2B cells. Each cover slip was incubated with 0.3 μl of CSC per 2 ml of media for indicated number of days. Protein aggregates were stained by Proteostat Aggresome Detection kit. B. NFI of the protein aggregates shown in A. C. Protein aggregation in A549 cells, incubated with 3 μl of CSC per 2 ml of media. Note that the amount of CSC used for A549 is 10 times more than for Beas-2B. D. NFI of the protein aggregates shown in C.
Fig 5
Fig 5. Comparison of the amounts of ABCG2 transcript in different cell lines.
The cells were incubated with or without 3 μl CSC per 2 ml of media for 1h, 3h, 6h, and 24 hours, or with 6 μl CSC for 24 hours. Amounts of ABCG2 were analyzed by real time RT-PCR. The amounts of ABCG2 transcript were normalized by the GAPDH transcript levels.
Fig 6
Fig 6. Increase of protein aggregation in A549 cells upon inhibition of ABCG2 with 1 μg/ml febuxostat.
A. Cells were treated with 0.3 μl of CSC per 2 ml of media or the same amount of DMSO. B. NFI of the protein aggregates shown in A.
Fig 7
Fig 7. Knockdown of ABCG2 in A549 resulted in increased protein aggregation.
A. CSC-induced protein aggregates in knockdown (shABCG2) and control (shNT, and A549) samples, stained by Proteostat Aggresome Detection kit. B. NFI of the protein aggregates shown in A. C. The real time RT-PCR with primers corresponding to ABCG2, showing the efficiency of the knockdown with or without CSC exposure for 24 hours; GAPDH was used for normalization.
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Grants and funding

This work was supported by a Tobacco Settlement (CURE) grant to Pennsylvania State University, Summer Undergraduate Research Experience (SURE) to E. S., and Undergraduate Research funding to E. P, A. S., E. S., M. S. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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