Bioenergetics of acquired cisplatin resistant H1299 non-small cell lung cancer and P31 mesothelioma cells

Our plain-English summary of Bioenergetics of acquired cisplatin resistant H1299 non-small cell lung cancer and P31 mesothelioma cells, by F. Geoghegan, R.J. Buckland, E.T. Rogers, et al (Oncotarget, 2017).

The study

• Authors: F. Geoghegan, R.J. Buckland, E.T. Rogers, K. Khalifa, E.B. O'Connor, M.F. Rooney, P. Behnam-Motlagh, T.K. Nilsson, K. Grankvist, R.K. Porter
• Journal: Oncotarget
• Published: 2017;8(55):94711-94725
• Cited by: 11 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Acquired cisplatin resistance is a common feature of tumours following cancer treatment with cisplatin and also of non-small cell lung cancer (H1299) and mesothelioma (P31) cell lines exposed to cisplatin. To elucidate the cellular basis of acquired cisplatin resistance, a comprehensive bioenergetic analysis was undertaken. We demonstrate that cellular oxygen consumption was significantly decreased in cisplatin resistant cells and that the reduction was primarily due to reduced mitochondrial activity as a result of reduced mitochondrial abundance. The differential mitochondrial abundance was supported by data showing reduced sirtuin 1 (SIRT1), peroxisome-proliferator activator receptor-γ co-activator 1-alpha (PGC1α), sirtuin 3 (SIRT3) and mitochondrial transcription factor A (TFAM) protein expression in resistant cells. Consistent with these data we observed increased reactive oxygen species (ROS) production and increased hypoxia inducible factor 1-alpha (HIF1α) stabilization in cisplatin resistant cells when compared to cisplatin sensitive controls. We also observed an increase in AMP kinase subunit α2 (AMPKα2) transcripts and protein expression in resistant H1299 cells. mRNA expression was also reduced for cisplatin resistant H1299 cells in these genes, however the pattern was not consistent in resistant P31 cells. There was very little change in DNA methylation of these genes, suggesting that the cells are not stably reprogrammed epigenetically. Taken together, our data demonstrate reduced oxidative metabolism, reduced mitochondrial abundance, potential for increased glycolytic flux and increased ROS production in acquired cisplatin resistant cells. This suggests that the metabolic changes are a result of reduced SIRT3 expression and increased HIF-1α stabilization.

In plain English

This laboratory study used cancer cells grown in culture and did not examine asbestos. The authors compared two cell lines, H1299 (non-small cell lung cancer) and P31 (from a patient with pleural malignant mesothelioma), with versions of each that had acquired resistance to the chemotherapy drug cisplatin. The cisplatin concentration needed to cut cell viability by half was 7.6 μmol/L for H1299 versus approx. 68.2 μmol/L for resistant H1299, and 5.8 versus 17.7 μmol/L for P31 versus resistant P31. Resistant cells grew more slowly, consumed less oxygen and had fewer mitochondria, the cell's energy-producing structures. Four proteins linked to building mitochondria were lower in resistant cells of both lines. Some tests were done only in the lung cancer line. There, resistant cells produced 2-fold more reactive oxygen species and showed more stabilized HIF1α, a protein that responds to low oxygen. DNA methylation of the metabolic control genes examined generally changed little. The authors concluded that the metabolic changes most likely result from reduced SIRT3 expression and increased HIF1α stabilization, and that they were partially evident in the mesothelioma cells.

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) cell-line experiment comparing cisplatin-resistant and parental cancer cells
• Who was studied: Two human cancer cell lines: H1299 (non-small cell lung cancer, from a lymph node metastasis) and P31 (from a male patient with a pleural malignant mesothelioma tumour), each compared with a cisplatin-resistant subline (H1299r, P31r)
• Exposure: Cisplatin (chemotherapy drug), not asbestos. Resistant sublines were maintained in cisplatin (2 mg/L for H1299r, 1.2 mg/L for P31r). Sensitivity tests used 50 nmol/L to 100 μmol/L for 72 h.
• Main results:
  • IC50 for cisplatin was 7.6 μmol/L in H1299 cells and approx. 68.2 μmol/L in H1299r cells (about 10-fold resistance)
  • IC50 for cisplatin was 5.8 μmol/L in parental P31 cells and 17.7 μmol/L in resistant P31r cells (about 3-fold resistance)
  • Parental cell lines proliferated about 2-fold faster than resistant cell lines (p less than 0.001)
  • Oxygen consumption was about 50% lower in H1299r than in H1299 cells under basal and all inhibitor/uncoupler conditions
  • In P31r compared with P31 cells, basal and FCCP-treated oxygen consumption were significantly lower, but oligomycin-treated consumption did not differ significantly
  • Citrate synthase activity, a marker of mitochondrial content, was about 2-fold greater in H1299 than H1299r cells (p less than 0.01) and about 1.5-fold greater in P31 than P31r cells (p less than 0.01)
  • Protein levels of SIRT1, PGC1α, SIRT3 and TFAM were reduced in resistant cells of both lines compared with parental cells
  • Extracellular acidification (index of glycolysis) was significantly lower in H1299r cells after oligomycin, FCCP and antimycin A. There was no significant difference between P31 and P31r cells.
  • H1299r cells produced 2-fold more reactive oxygen species than H1299 cells
  • Stabilized HIF1α expression was increased in H1299r compared with H1299 cells. It was tested in H1299 cells only, not P31.
  • AMPKα2 protein showed a significant 3-fold increase in H1299r cells compared with parental cells
  • DNA methylation of the four mitochondrial biogenesis genes showed only a slight decrease or no change in both cell lines
  • Overall, methylation changed little in the metabolic control genes studied in either cell line. One exception was PRKAA2 (the gene for AMPKα2) in P31 cells, with a larger methylation change of +0.2 beta despite very little mRNA change.
• The authors' conclusion: The authors concluded that reduced oxidative metabolism, reduced mitochondrial abundance and increased ROS in resistant cells most likely result from reduced SIRT3 expression and increased HIF1α stabilization, and that these changes are partially evident in P31 mesothelioma cells
• Limitations the authors state:
  • Extracellular acidification rate may not truly reflect glycolytic rate, because carbon dioxide production also acidifies the medium
  • The uncoupler FCCP may have secondary effects, so maximal oxidative capacity may be underestimated
  • The relevance of the increased AMPKα2 subunit to cisplatin resistance is not clear

Topics

Medical terms in this study, explained in our Lexicon: Cisplatin, Non-Small Cell Lung Cancer, Mesothelioma, Reactive Oxygen Species (ROS), Hypoxia.

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• Randomised phase II study of cisplatin-etoposide versus infusional carboplatin in advanced non-small-cell lung cancer and mesothelioma — S.C. White, H. Anderson, et al. (2000)
• Sunitinib combined with pemetrexed and cisplatin: results of a phase I dose-escalation and pharmacokinetic study in patients with advanced solid malignancies, with an expanded cohort in non-small cell lung cancer and mesothelioma — D.R. Camidge, N. Blais, et al. (2013)

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