Molecular mechanism implicated in Pemetrexed-induced apoptosis in human melanoma cells

Our plain-English summary of Molecular mechanism implicated in Pemetrexed-induced apoptosis in human melanoma cells, by A. Buqué, J.S.H. Muhialdin, A. Muñoz, et al (Molecular Cancer, 2012).

The study

• Authors: A. Buqué, J.S.H. Muhialdin, A. Muñoz, B. Calvo, S. Carrera, U. Aresti, A. Sancho, I. Rubio, G. López-Vivanco
• Journal: Molecular Cancer
• Published: 2012;11:25
• Cited by: 40 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Background: Metastatic melanoma is a lethal skin cancer and its incidence is rising every year. It represents a challenge for oncologist, as the current treatment options are non-curative in the majority of cases; therefore, the effort to find and/or develop novel compounds is mandatory. Pemetrexed (Alimta®, MTA) is a multitarget antifolate that inhibits folate-dependent enzymes: thymidylate synthase, dihydrofolate reductase and glycinamide ribonucleotide formyltransferase, required for de novo synthesis of nucleotides for DNA replication. It is currently used in the treatment of mesothelioma and non-small cell lung cancer (NSCLC), and has shown clinical activity in other tumors such as breast, colorectal, bladder, cervical, gastric and pancreatic cancer. However, its effect in human melanoma has not been studied yet.

Results: In the current work we studied the effect of MTA on four human melanoma cell lines A375, Hs294T, HT144 and MeWo and in two NSCLC cell lines H1299 and Calu-3. We have found that MTA induces DNA damage, S-phase cell cycle arrest, and caspase- dependent and -independent apoptosis. We show that an increment of the intracellular reactive oxygen species (ROS) and p53 is required for MTA-induced cytotoxicity by utilizing N-Acetyl-L-Cysteine (NAC) to blockage of ROS and p53-defective H1299 NSCLC cell line. Pretreatment of melanoma cells with NAC significantly decreased the DNA damage, p53 up-regulation and cytotoxic effect of MTA. MTA was able to induce p53 expression leading to up-regulation of p53-dependent genes Mcl-1 and PIDD, followed by a postranscriptional regulation of Mcl-1 improving apoptosis.

Conclusions: We found that MTA induced DNA damage and mitochondrial-mediated apoptosis in human melanoma cells in vitro and that the associated apoptosis was both caspase-dependent and -independent and p53-mediated. Our data suggest that MTA may be of therapeutic relevance for the future treatment of human malignant melanoma.

In plain English

This was a laboratory study of cancer cells grown in dishes. It did not study people or asbestos exposure. The authors tested pemetrexed (MTA), a chemotherapy drug the article says is used to treat mesothelioma and non-small cell lung cancer. They tested it on four human melanoma cell lines (A375, Hs294T, HT144 and MeWo), two lung cancer cell lines (Calu-3 and H1299) and mouse embryo fibroblasts. Pemetrexed reduced survival of the melanoma cells and Calu-3 cells. The article reported no significant cytotoxic effect on the fibroblasts or on H1299, a line lacking the p53 protein. The concentrations that halved viability in the melanoma lines were 0.27 to 0.99 micromolar. In melanoma cells the drug caused DNA damage, an arrest of the cell cycle in the S phase, higher reactive oxygen species, and programmed cell death involving mitochondria and caspase enzymes. Blocking reactive oxygen species with N-acetyl-cysteine reversed the drug's cytotoxicity and reduced DNA damage and p53 expression. The authors concluded that pemetrexed may be of therapeutic relevance for future melanoma treatment. They noted that additional pathways may be involved.

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 experiment on cultured cell lines
• Who was studied: Four human melanoma cell lines (A375, Hs294T, HT144, MeWo) and two non-small cell lung cancer cell lines (Calu-3, H1299), obtained from the American Type Culture Collection, plus primary mouse embryo fibroblasts as non-tumor cells. No human subjects were studied.
• Exposure: Pemetrexed (MTA) at various concentrations (0.17 to 10 micromolar) for various periods (24 to 72 h). In some experiments, 10 mM N-acetyl-cysteine was added 1 h before MTA to block reactive oxygen species. No asbestos exposure was studied.
• Main results:
  • MTA significantly reduced cell viability in all four melanoma cell lines and in Calu-3. There was no significant effect on H1299, which lacks p53 expression.
  • There was no significant cytotoxic effect on mouse embryo fibroblasts
  • The concentrations of MTA that inhibited viability by 50% were 0.27, 0.41, 0.32 and 0.99 micromolar for A375, Hs294T, HT144 and MeWo melanoma cells, and 10 micromolar for Calu-3.
  • After 24 h exposure to 0.84 micromolar MTA, colony-forming ability was significantly reduced in the melanoma cells and in Calu-3 cells, but not in H1299
  • In melanoma cells, 0.84 micromolar MTA increased the percentage of hypodiploid (apoptotic) cells at 24, 48 and 72 h. A lower dose of 0.17 micromolar MTA led to a transient arrest in the S phase of the cell cycle.
  • The comet assay showed DNA strand breaks in melanoma cells after early MTA exposure
  • In Hs294T cells, a general (pan-caspase) inhibitor reduced the MTA effect significantly but not totally. Inhibitors of caspase-2, -3, -9 and -10 were the most effective, while the caspase-8 inhibitor did not significantly reverse the MTA effect.
  • Intracellular reactive oxygen species rose significantly after 18 h of MTA treatment. Pretreatment of melanoma cells with N-acetyl-cysteine reversed cytotoxicity and reduced DNA damage and p53 expression.
  • In HT144 cells, blocking p53 activity with pifithrin-alpha prevented the MTA-induced S-phase arrest (cells in S phase: control 16.16%, MTA 30.62%, PFT plus MTA 17%). No significant reduction of cells in the sub-G0/G1 (dying) fraction was detected when p53 activity was inhibited.
  • MTA caused loss of mitochondrial membrane potential. Cytochrome c and AIF decreased in the mitochondrial fraction and increased in the cytosol of treated cells.
  • MTA raised mRNA levels of PUMA, PIDD and Mcl-1. Silencing p53 reduced the PIDD and Mcl-1 increases but not the PUMA increase. PUMA protein did not change, and Mcl-1 protein decreased after MTA treatment.
• The authors' conclusion: The authors concluded that MTA induced DNA damage and mitochondrial-mediated apoptosis in human melanoma cells in vitro, and that this apoptosis was caspase-dependent and -independent and p53-mediated. They suggested MTA may be of therapeutic relevance for future treatment of human malignant melanoma and that their results should encourage clinical trials.
• Limitations the authors state:
  • The authors stated that additional pathways, not identified in this study, may also contribute to MTA-induced apoptosis
  • The authors stated that the exact mechanism of caspase-2 and caspase-10 activation remains unclear

Topics

Medical terms in this study, explained in our Lexicon: Pemetrexed (Alimta), Apoptosis, Antifolate, De Novo, Mesothelioma, Non-Small Cell Lung Cancer, Reactive Oxygen Species (ROS).

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• Usp9x- and Noxa-mediated Mcl-1 downregulation contributes to pemetrexed-induced apoptosis in human non-small-cell lung cancer cells — J. Yan, N. Zhong, et al. (2014)
• Death receptor 5 and cellular FLICE-inhibitory protein regulate pemetrexed-induced apoptosis in human lung cancer cells — L. Su, G. Liu, et al. (2011)
• Enhanced apoptosis by pemetrexed and simvastatin in malignant mesothelioma and lung cancer cells by reactive oxygen species-dependent mitochondrial dysfunction and Bim induction — K.E. Hwang, Y.S. Kim, et al. (2014)
• Preclinical emergence of vandetanib as a potent antitumour agent in mesothelioma: molecular mechanisms underlying its synergistic interaction with pemetrexed and carboplatin — E. Giovannetti, P.A. Zucali, et al. (2011)
• Interferon-β produces synergistic combinatory anti-tumor effects with cisplatin or pemetrexed on mesothelioma cells — Q. Li, K. Kawamura, et al. (2013)

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