Elsevier

Toxicology in Vitro

Volume 25, Issue 8, December 2011, Pages 1568-1574
Toxicology in Vitro

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

https://doi.org/10.1016/j.tiv.2011.05.023Get rights and content

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.

Highlights

Gemcitabine and epigallocatechin-3-gallate synergize with ascorbate on mesothelioma. ► Gemcitabine/ascorbate is a promising combined therapy for malignant mesothelioma. ► Epigallocatechin-3-gallate/ascorbate is another promising therapy for mesothelioma.

Introduction

Malignant mesothelioma (MMe) is a lethal tumor arising from the mesothelium of serous cavities, whose incidence has been increasing in several countries as a result of widespread use of asbestos (Carbone et al., 2002). In the United States of America the incidence of MMe reached its peak in the early to mid 1990s and is now on a downward trend. However, in Europe a peak of incidence is expected around 2020, while a still long-lasting trend is forecasted for some Asian countries and Australia (Weill et al., 2004, Peto et al., 1999, Connelly et al., 1987).

MMe can be classified into epithelial, sarcomatoid or biphasic (containing both epithelial and sarcomatoid elements) histological subtypes. The prognosis of these tumors is very poor, with a median survival of 9 months from diagnosis (Ceresoli et al., 2001). An effective therapy for MMe has not been established yet: surgery is very invasive and radiotherapy is not a radical treatment, while more encouraging results have been obtained with chemotherapy. The current standard for the systemic treatment of advanced MMe is a combination of cisplatin and pemetrexed or raltitrexed. However, currently there is no widely approved salvage regimen after failure of first-line treatment. In view of the poor survival benefit from first-line chemotherapy, and the lack of subsequent effective treatment options, there is a need for the development of new more effective curative modalities.

Ascorbate, also known as vitamin C, has been considered since long as a remedy in the treatment of cancer (Cameron and Pauling, 1978). Thereafter, a number of studies have been dedicated to the anticancer properties of ascorbate. Antitumor effects have been observed against a wide variety of cancer cells both in vitro and in preclinical models (Verrax and Calderon, 2009, Du et al., 2010, Takemura et al., 2010, Pollard et al., 2010, Deubzer et al., 2010). Moreover, evidence for an effectiveness of ascorbate treatments has also emerged from clinical experience (Riordan et al., 2004, Riordan et al., 2005, Casciari et al., 2005). We have previously demonstrated that the compound is selectively cytotoxic toward MMe cells, showing that ascorbate-induced extracellular H2O2 production induces a strong oxidative stress in MMe cells due to their high superoxide production rate (Ranzato et al., 2011). The elucidation of this mechanism explains the selective toxicity of ascorbate to MMe cells, and suggests its possible use in the clinical treatment of mesothelioma.

Clinical data have generally shown that MMe is a particularly chemoresistant tumor, and in agreement with this kind of evidence, best results in the medical treatment of the disease have been obtained with combinations of different compounds (Fennell et al., 2008). Hence, by considering that at concentrations that are toxic for MMe cells (Ranzato et al., 2011) ascorbate is well tolerated by the human body (Riordan et al., 2005), we have attempted at finding partner compounds which could act synergistically with ascorbate against MMe cell growth. We have therefore tested common chemotherapeutic drugs used in MMe therapy, such as cisplatin, etoposide, gemcitabine, imatinib, paclitaxel, and raltitrexed (Belli et al., 2009). Moreover, due to the stringent need of novelties in MMe therapy, we have also included in our screening a few compounds, which have not been used yet in clinical settings on MMe, but are considered promising antitumor agents. These latter include taurolidine, a pro-apoptotic and anti-angiogenic drug already tested in vitro on MMe cells (Aceto et al., 2009), and (+)-α-tocopherol succinate, a vitamin E analog that has been found to arrest cancer cell proliferation (Constantinou et al., 2008). Finally, epigallocatechin-3-gallate (EGCG), a natural anti-cancer compound present in green tea (Azam et al., 2004, Chen et al., 2011) has been also tested in combination with ascorbate, due to its reported tendency to interact synergistically with different anticancer drugs (Suganuma et al., 2011).

Isobologram analyses, based on in vitro cytotoxicity tests, have shown synergistic interactions of ascorbate with EGCG and gemcitabine. These results have been confirmed through inferential and combination-index analysis, and also by using other endpoints, i.e. lactic dehydrogenase release and caspase-3 activation. Hence, the complex of data suggests that these mixtures could be exploited in the clinical treatment of MMe.

Section snippets

Reagents and solutions

(−)-Epigallocatechin-3-gallate (EGCG) was purchased from Cayman Chemical Co. (Ann Arbor, MI, USA); gemcitabine (2′-deoxy-,2′-difluorocytidine monohydrochloride, Gemzar) was from Ely Lilly Italia S.p.A. (Sesto Fiorentino, Italy); taurolidine (4,4′-methylenebis(tetrahydro-1,2,4-sulfadiazine 1,1-dioxide) and tyrphostin AG 1478 (N-(3-chlorophenyl)-6,7-dimethoxy-4-quinazolinamine) were from Alexis Biochemicals (Lausen, Switzerland). l-ascorbic acid, cisplatin (cis-diamineplatinum(II) dichloride),

Cytotoxicity tests for single compounds

The NRU analysis of MMe REN cells exposed to graded concentrations of the different compounds allowed us to obtain for each compound a cell viability-inhibition, dose–response curve (Fig. 1), yielding a median effective concentration (IC50) (Table 2). As found in our previous study (Ranzato et al., 2011) the IC50 of ascorbate was almost in the millimolar range. Conversely, all the other IC50s were in the mid-to-low micromolar range, with the exception of raltitrexed, whose toxicity, at 24, 48,

Discussion

In the therapy of MMe, low response rates to various single chemotherapy agents have been always obtained (Ellis et al., 2006), suggesting the need for improved chemotherapy efficacy and/or selection to optimize multimodality treatments. Although combination chemotherapy has been associated with higher response rates in patients, these results do not necessarily translate into longer median survival (Kalmadi et al., 2008).

The combinations of cisplatin and pemetrexed or raltitrexed were

Conflict of interest

I declare that none of the authors has actual or potential conflict of interest.

Acknowledgements

This work was supported by Fondazione Buzzi Unicem, Casale Monferrato, Italy. ER is recipient of a Research Fellowship from the University of Piemonte Orientale. SM is recipient of a PhD scholarship from the Italian Ministry of University and Research (MIUR).

References (50)

  • J.M. Grad et al.

    Mitochondria as targets for established and novel anti-cancer agents

    Drug Resist. Updat.

    (2001)
  • G. Hillerdal et al.

    Treatment of malignant pleural mesothelioma with carboplatin, liposomized doxorubicin, and gemcitabine: a phase II study

    J Thorac. Oncol.

    (2008)
  • L.J. Hoffer et al.

    Phase I clinical trial of i.v. ascorbic acid in advanced malignancy

    Ann. Oncol.

    (2008)
  • S.R. Kalmadi et al.

    Gemcitabine and cisplatin in unresectable malignant mesothelioma of the pleura: a phase II study of the Southwest Oncology Group (SWOG 9810)

    Lung Cancer

    (2008)
  • C. Legrand et al.

    Lactate dehydrogenase (LDH) activity of the cultured eukaryotic cells as marker of the number of dead cells in the medium

    J. Biotechnol.

    (1992)
  • M. Pistolesi et al.

    Malignant pleural mesothelioma: update, current management, and newer therapeutic strategies

    Chest

    (2004)
  • Y. Takemura et al.

    High dose of ascorbic acid induces cell death in mesothelioma cells

    Biochem. Biophys. Res. Commun.

    (2010)
  • R.J. Tallarida et al.

    Statistical analysis of drug–drug and site–site interactions with isobolograms

    Life Sci.

    (1989)
  • J. Verrax et al.

    Pharmacologic concentrations of ascorbate are achieved by parenteral administration and exhibit antitumoral effects

    Free Radic. Biol. Med.

    (2009)
  • N. Aceto et al.

    Taurolidine and oxidative stress: a rationale for local treatment of mesothelioma

    Eur. Respir. J.

    (2009)
  • Barnes, M., Correll, R., Stevens, D., 2003. A simple spreadsheet for estimating low-effect concentrations and...
  • C. Belli et al.

    Malignant pleural mesothelioma: current treatments and emerging drugs

    Expert. Opin. Emerg. Drugs

    (2009)
  • E. Cameron et al.

    Supplemental ascorbate in the supportive treatment of cancer: reevaluation of prolongation of survival times in terminal human cancer

    Proc. Natl. Acad. Sci. USA

    (1978)
  • J.J. Casciari et al.

    Effects of high dose ascorbate administration on L-10 tumor growth in guinea pigs

    PR Health Sci. J.

    (2005)
  • Q. Chen et al.

    Pharmacologic doses of ascorbate act as a prooxidant and decrease growth of aggressive tumor xenografts in mice

    Proc. Natl. Acad. Sci. USA

    (2008)
  • Cited by (0)

    View full text