In vitro screening of synergistic ascorbate-drug combinations for the treatment of malignant mesothelioma

Our plain-English summary of In vitro screening of synergistic ascorbate-drug combinations for the treatment of malignant mesothelioma, by S. Martinotti, E. Ranzato, B. Burlando (Toxicology in Vitro, 2011).

The study

• Authors: S. Martinotti, E. Ranzato, B. Burlando
• Journal: Toxicology in Vitro
• Published: 2011;25(8):1568-74
• Cited by: 29 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

Malignant mesothelioma (MMe) is a lethal tumor arising from the mesothelium of serous cavities as a result of exposure to asbestos. Current clinical standards consist of combined treatments, but an effective therapy has not been established yet and there is an urgent need for new curative approaches. Ascorbate is a nutrient that is also known as a remedy in the treatment of cancer. In the present study, we have tested the cytotoxicity of ascorbate to MMe cells in combination with drugs used in MMe therapy, such as cisplatin, etoposide, gemcitabine, imatinib, paclitaxel, and raltitrexed, as well as with promising antitumor compounds like taurolidine, α-tocopherol succinate, and epigallocatechin-3-gallate (EGCG). Dose-response curves obtained for each compound by applying the neutral red uptake (NRU) assay to MMe cells growing in vitro, allowed to obtain IC50 values for each compound used singularly. Thereafter, NRU data obtained from each ascorbate/drug combination were analyzed through Tallarida's isobolograms at the IC50 level (Tallarida, 2000), revealing synergistic interactions for ascorbate/gemcitabine and ascorbate/EGCG. These results were further confirmed through comparisons between theoretical additivity IC50 and observed IC50 from fixed-ratio dose-response curves, and over a broad range of IC levels, by using Chou and Talalay's combination index (Chou and Talalay, 1984). Synergistic interactions were also shown by examining apoptosis and necrosis rates, using the caspase 3 and lactic dehydrogenase assays, respectively. Hence, data indicate that ascorbate/gemcitabine and ascorbate/EGCG affect synergistically the viability of MMe cells and suggest their possible use in the clinical treatment of this problematic cancer.

Copyright © 2011 Elsevier Ltd. All rights reserved.

In plain English

In this laboratory study, the authors treated one malignant mesothelioma cell line, REN, grown in culture; no patients, animals or asbestos exposure were studied. They measured cell survival after ascorbate (vitamin C) alone, several drugs alone, and ascorbate paired with each drug. Ascorbate needed concentrations close to the millimolar range to halve survival, while most drugs worked at micromolar levels. Raltitrexed was too weakly toxic for that concentration to be estimated, so it was dropped. At the halving level, ascorbate with gemcitabine or with EGCG (a green tea compound) reduced survival more than their separate effects added together, which the authors called synergy. At 48 hours, though, ascorbate with EGCG was antagonistic at the lowest inhibition levels and synergistic only from 40% upward; ascorbate with gemcitabine was synergistic at all levels. Pairings with cisplatin were additive; several others, including etoposide and paclitaxel, were antagonistic. The two synergistic pairs also raised necrosis and apoptosis markers more than single compounds. The authors concluded that these combinations act synergistically against mesothelioma cell proliferation and that ascorbate deserves serious consideration in combination treatment.

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-culture study testing drug combinations for toxicity to mesothelioma cells, using isobologram and combination-index analysis
• Who was studied: A single human malignant mesothelioma cell line, REN, described as a p53-mutant epithelial line, grown in culture. No patients or animals were studied.
• Exposure: Laboratory drug treatments, not asbestos. The cells received ascorbate alone, each comparison compound alone, and ascorbate combined with each compound, for 24, 48 or 72 hours. The comparison compounds were cisplatin, etoposide, gemcitabine, imatinib, paclitaxel, raltitrexed, taurolidine, alpha-tocopherol succinate, epigallocatechin-3-gallate (EGCG) and tyrphostin AG 1478.
• Main results:
  • Used alone on REN cells, ascorbate had a 50% inhibitory concentration (IC50) almost in the millimolar range: 1097 micromolar at 24 h (95% CI 1036 to 1161) and 820 micromolar at 48 h (95% CI 750 to 890). The other compounds had IC50s in the mid-to-low micromolar range, except raltitrexed.
  • At 24, 48 or 72 h, raltitrexed's toxicity was too low for an IC50 to be estimated, so it was excluded from the combination tests.
  • Isobologram analysis at the IC50 level showed synergism for ascorbate combined with gemcitabine and for ascorbate combined with EGCG.
  • The same analysis showed strong antagonism for ascorbate with imatinib, etoposide and taurolidine, and weaker antagonism with paclitaxel and tocopherol. Ascorbate with cisplatin or with tyrphostin was additive.
  • In fixed-ratio tests, the observed IC50s of the combinations were significantly lower than the IC50s expected if the effects were simply additive. Ascorbate plus gemcitabine at 48 h: expected 444, observed 170 (95% CI 144 to 201) micromolar. Ascorbate plus EGCG at 24 h: expected 462, observed 225 (170 to 298). Ascorbate plus EGCG at 48 h: expected 425, observed 166 (142 to 193).
  • Combination-index analysis showed synergism for ascorbate plus gemcitabine at all levels of cell-viability inhibition tested.
  • Ascorbate plus EGCG gave results similar to ascorbate plus gemcitabine at 24 h. At 48 h it was antagonistic at the lowest inhibition levels, additive at an inhibition fraction of 0.3, and synergistic from 0.4 upward.
  • After 6 h, ascorbate plus EGCG produced a significantly higher release of lactic dehydrogenase (a marker of cell necrosis) than either compound alone. Ascorbate plus gemcitabine produced an even higher release. In the combinations each compound was used at half its IC50; alone, each was used at its full IC50.
  • Similar differences between the single compounds and the combinations were found for caspase 3 activity (a marker of apoptosis) after 6 h of treatment.
• The authors' conclusion: The authors concluded that gemcitabine and EGCG, each combined with ascorbate, act synergistically to inhibit mesothelioma cell proliferation. They also concluded that ascorbate should be seriously considered when developing combination treatments for malignant mesothelioma.
• Limitations the authors state:
  • The authors noted that the effect of EGCG and other green tea polyphenols on the growth of mesothelioma tumors in living organisms is unknown.

Topics

The National Library of Medicine indexes this study under subjects our site covers: Lung cancer, Mesothelioma.

Medical terms in this study, explained in our Lexicon: Malignant Mesothelioma, Mesothelium, Serous Cavities, Cisplatin, Gemcitabine, Raltitrexed, Dose-response relationship, Apoptosis.

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