Augmented expression of cardiac ankyrin repeat protein is induced by pemetrexed and a possible marker for the pemetrexed resistance in mesothelioma cells

Our plain-English summary of Augmented expression of cardiac ankyrin repeat protein is induced by pemetrexed and a possible marker for the pemetrexed resistance in mesothelioma cells, by Y. Qin, I. Sekine, M. Fan, et al (Cancer Cell International, 2017).

The study

• Authors: Y. Qin, I. Sekine, M. Fan, Y. Takiguchi, Y. Tada, M. Shingyoji, M. Hanazono, N. Yamaguchi, M. Tagawa
• Journal: Cancer Cell International
• Published: 2017;17:120
• Cited by: 1 article, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Background: Pemetrexed (PEM) is an anti-cancer agent targeting DNA and RNA synthesis, and clinically in use for mesothelioma and non-small cell lung carcinoma. A mechanism of resistance to PEM is associated with elevated activities of several enzymes involved in nucleic acid metabolism.

Methods: We established two kinds of PEM-resistant mesothelioma cells which did not show any increase of the relevant enzyme activities. We screened genes enhanced in the PEM-resistant cells with a microarray analysis and confirmed the expression levels with Western blot analysis. A possible involvement of the candidates in the PEM-resistance was examined with a WST assay after knocking down the expression with si-RNA. We also analyzed a mechanism of the up-regulated expression with agents influencing AMP-activated protein kinase (AMPK) and p53.

Results: We found that expression of cardiac ankyrin repeat protein (CARP) was elevated in the PEM-resistant cells with a microarray and Western blot analysis. Down-regulation of CARP expression with si-RNA did not however influence the PEM resistance. Parent and PEM-resistant cells treated with PEM increased expression of CARP, AMPK, p53 and histone H2AX. The CARP up-regulation was however irrelevant to the p53 genotypes and not induced by an AMPK activator. Augmented p53 levels with nutlin-3a, an inhibitor for p53 degradation, and DNA damages were not always associated with the enhanced CARP expression.

Conclusions: These data collectively suggest that up-regulated CARP expression is a potential marker for development of PEM-resistance in mesothelioma and that the PEM-mediated enhanced expression is not directly linked with immediate cellular responses to PEM.

In plain English

This laboratory study used human mesothelioma cell lines, not patients, and did not examine asbestos exposure. It looked at pemetrexed, a chemotherapy drug used for mesothelioma, and at cells that resist it. The authors compared four mesothelioma cell lines with drug-resistant versions made earlier by exposing them to rising pemetrexed doses. A gene screen of two parent-and-resistant pairs found six genes whose expression was more than 5 times higher in the resistant cells than in the parent cells. One was cardiac ankyrin repeat protein (CARP). CARP protein was raised in the resistant cells except one line (H2452-PEM). Lowering CARP with si-RNA, a gene-silencing tool, did not change the cells' sensitivity to pemetrexed. A second candidate, IGFBP3, was found not to be involved. Pemetrexed treatment itself raised CARP in the main parent and resistant cells tested. Further tests indicated this rise was not directly dependent on DNA damage or on activation of the enzyme AMPK. Its link to the p53 pathway depended on cell type. The authors suggested raised CARP is a candidate marker for pemetrexed resistance in mesothelioma. They noted such a marker is not clinically useful at present.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: laboratory (in vitro) study of human cell lines
• Who was studied: Human mesothelioma cell lines NCI-H28, NCI-H226, MSTO-211H and NCI-H2452 and their pemetrexed-resistant versions (H28-PEM, H226-PEM, 211H-PEM, H2452-PEM); mesothelioma lines EHMES-1 and JMN-1B (mutated p53); immortalized mesothelium-derived Met-5A cells
• Exposure: The chemotherapy drug pemetrexed (PEM), plus nutlin-3a (raises p53) and A769662 (activates AMPK). No asbestos or fiber exposure was studied.
• Main results:
  • All four pemetrexed-resistant cell lines were less sensitive to pemetrexed than their parent cells in the WST viability assay
  • Microarray screening found six genes whose expression was more than 5 times higher in the resistant cells than in the parent cells, in the two pairs NCI-H28/H28-PEM and NCI-H226/H226-PEM. For CARP the ratios were 6.320 (H28-PEM/NCI-H28) and 28.949 (H226-PEM/NCI-H226).
  • On Western blot, CARP protein was increased in all the pemetrexed-resistant cell lines except H2452-PEM
  • Lowering CARP with three different si-RNAs in H28-PEM cells did not change their sensitivity to pemetrexed
  • Experiments with IGFBP3, another candidate gene, indicated it was not involved in pemetrexed sensitivity
  • Pemetrexed treatment increased CARP but not IGFBP3 expression in the NCI-H28 and NCI-H226 parent and resistant cells
  • In cells with mutated or non-functional p53, CARP rose in EHMES-1 but fell in JMN-1B and Met-5A, while a DNA damage marker rose in all of them. The authors took this to mean the CARP increase was not directly dependent on DNA damage.
  • The AMPK activator A769662 raised phosphorylated AMPK but did not induce CARP
  • Raising p53 with nutlin-3a increased CARP in NCI-H28 and H28-PEM cells but generally not in NCI-H226-derived cells. The authors described the role of the p53 pathway as cell-type dependent.
• The authors' conclusion: The authors concluded that raised CARP expression is a candidate marker for pemetrexed resistance in mesothelioma, even though lowering CARP did not affect pemetrexed sensitivity. They also concluded that activation of the p53 pathway was involved in the CARP increase depending on cell type.
• Limitations the authors state:
  • The authors did not know why mRNA and protein levels of the candidate genes disagreed in the paired cells
  • The mechanism of resistance in the established resistant cells was not well characterized
  • The precise mechanism of the CARP increase remained unclear, and the increase was not related to pemetrexed resistance
  • A pemetrexed-resistance marker is not clinically useful at present, and tumor samples from patients who fail to respond are usually unavailable

Topics

Medical terms in this study, explained in our Lexicon: Pemetrexed (Alimta), Mesothelioma, Carcinoma, Damages.

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