In vitro studies on cisplatin focusing on kinetic aspects of intracellular chemistry by LC-ICP-MS

Our plain-English summary of In vitro studies on cisplatin focusing on kinetic aspects of intracellular chemistry by LC-ICP-MS, by G. Hermann, P. Heffeter, T. Falta, et al (Metallomics, 2013).

The study

• Authors: G. Hermann, P. Heffeter, T. Falta, W. Berger, S. Hann, G. Koellensperger
• Journal: Metallomics
• Published: 2013;5(6):636-47
• Cited by: 23 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

To date, preclinical studies have addressed drug accumulation and intracellular distribution of cisplatin by determination of the total Pt content. In this work, the use of liquid chromatography in combination with inductively coupled plasma mass spectrometry (LC-ICP-MS) enabled accurate intact cisplatin quantification in cell model experiments. Hence, for the first time, intracellular drug degradation, drug accumulation and drug efflux were studied by actually quantifying the intact drug, along with the total Pt content of the cell nucleus, the cytosol and the low molecular weight fraction of the cytosol. The latter fraction was obtained by centrifugal filtration (cut-off filter of 10 kDa). Flow injection (FI)-ICP-MS was implemented for platinum quantification. In a first step, kinetics of intracellular cisplatin degradation was addressed by incubating cell extracts with sub-μM drug concentration levels. A half-life of 2 hours was observed in cell extracts of two different cancer cell lines (colon carcinoma and human mesothelioma), which was significantly shorter than that observed in sodium chloride. Hence, it was suggested that intact and nonaquated cisplatin was reacting with cellular components. Due to the large excess of potential binding partners pseudo first order kinetics were observed. The drug accumulation experiments revealed rapid uptake of the drug into the cytosol and the nucleus. Moreover, a significant fraction of Pt was bound to intracellular high molecular weight biomolecules after one hour of exposure. With ongoing time, the intracellular Pt concentration was increasing. However, the cisplatin concentration remained constant during 5 hours of continuous exposure. Assuming a cell volume of 10(-12) L, an intracellular concentration corresponding to the cisplatin concentration in the cell culture medium (5 μM) was estimated. At any time of investigation, intact cisplatin was the predominant species in the low molecular weight fraction of the cytosol. These findings support the hypothesis of passive diffusion as an uptake mechanism. Finally, a model experiment was designed resembling the situation of limited drug exposure time. Human mesothelioma cells were incubated with 5 μM cisplatin for 3 hours. Then the culture medium was replaced and the drug efflux was studied. The observed efflux was biphasic, with the intact cisplatin being removed within the first hour of investigation, while the Pt-protein adduct fraction was removed only partially (30% were still found in the cytosol after 24 hours). No net transfer of Pt from the cytosol to the nucleus fraction was observed after medium replacement.

In plain English

This laboratory study did not examine asbestos exposure. It studied the cancer drug cisplatin in extracts of a human mesothelioma line (P31) and a colon carcinoma line (HCT116), and in living P31 cells only. Mass-spectrometry methods measured intact cisplatin separately from total platinum. When cisplatin was added to these extracts, its half-life was about 2 hours. In 5 mM and 15 mM sodium chloride solutions it was 8.4 hours. After 2 hours, 40% of the platinum in HCT116 extracts and 28% in P31 extracts was bound to large molecules such as proteins. In P31 cells exposed to 5 micromolar cisplatin for 5 hours, 2% of the drug offered became cell-associated, and platinum reached the nucleus within one hour. In P31 cells exposed for 3 hours and then given drug-free medium, intact cisplatin was almost completely removed within the first hour. Protein-bound platinum fell only to 30 to 40% of its starting level by 24 hours. The authors concluded that diffusion was the main way cisplatin entered the cells, and that binding to thiols was highly likely.

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: In vitro laboratory experiments on human cancer cell lines and cell extracts
• Who was studied: Two human cancer cell lines: the mesothelioma cell model P31 (donated by K. Grankvist, Umeå University, Sweden) and the colon carcinoma cell model HCT116 (from B. Vogelstein, Johns Hopkins). The cell-uptake and drug-removal experiments used P31 cells only.
• Exposure: The platinum chemotherapy drug cisplatin, not asbestos. Cells were exposed at 5 micromolar for up to 5 hours, or for 3 hours followed by drug-free medium. Cell extracts were spiked with 100 micrograms per litre (332 nM).
• Main results:
  • In cell extracts (HCT116 and P31), cisplatin had a half-life of about 2 hours.
  • In 5 mM and 15 mM sodium chloride solutions, the cisplatin half-life was 8.4 hours (text figure).
  • The two cell lines had significantly different glutathione levels but a comparable cisplatin half-life.
  • In HCT116 cell extracts, after 2 hours 40% of total platinum was in the high molecular weight fraction (above 10 kDa).
  • In P31 cell extracts, after 2 hours 28% of platinum was in the high molecular weight fraction.
  • The authors inferred from the decay experiments that intact cisplatin formed adducts before hydrolysis, and that thiol binding was highly likely.
  • In P31 cells continuously exposed for 5 hours, 2% of the offered cisplatin was cell-associated.
  • In P31 cells, a significant fraction of platinum was found in the nucleus after one hour of exposure.
  • In P31 cells, one hour after drug-containing medium was replaced, cell-associated platinum had decreased by 60%.
  • In P31 cells, intact cisplatin was nearly completely removed from the cytosol within the first hour after medium replacement.
  • In P31 cells, platinum bound to high molecular weight biomolecules fell only to 30 to 40% of its initial level within 24 hours.
• The authors' conclusion: The authors concluded that rapid diffusion was the predominant way cisplatin entered the cells.
• Limitations the authors state:
  • The half-life estimate for P31 cell extracts was more uncertain because it was based on only 3 time points.

Topics

Medical terms in this study, explained in our Lexicon: Cisplatin, Carcinoma, Mesothelioma, Biphasic Mesothelioma.

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