Zoledronic acid produces antitumor effects on mesothelioma through apoptosis and S-phase arrest in p53-independent and Ras prenylation-independent manners

Our plain-English summary of Zoledronic acid produces antitumor effects on mesothelioma through apoptosis and S-phase arrest in p53-independent and Ras prenylation-independent manners, by S. Okamoto, K. Kawamura, Q. Li, et al (Journal of Thoracic Oncology, 2012).

The study

• Authors: S. Okamoto, K. Kawamura, Q. Li, M. Yamanaka, S. Yang, T. Fukamachi, Y. Tada, K. Tatsumi, H. Shimada, K. Hiroshima, H. Kobayashi, M. Tagawa
• Journal: Journal of Thoracic Oncology
• Published: 2012;7(5):873-82
• Cited by: 23 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

Introduction: We examined whether zoledronic acid (ZOL), the third generation of bisphosphonates, produced cytotoxic effects on human mesothelioma cells in vitro and in vivo, and investigated a possible involvement of p53, Ras, and extracellular signal-regulated kinase1/2 (ERK1/2) pathways.

Methods: Cytotoxicity and cell cycles were assessed with a colorimetric assay and flow cytometry, respectively. Expression levels of apoptosis-linked proteins and prenylation of small guanine-nucleotide-binding regulatory proteins were tested with p53-small interfering RNA, an ERK kinase1/2-inhibitor, and prenyl alcohols. The antitumor activity was examined in an orthotopic animal model.

Results: ZOL treatments suppressed growth of mesothelioma cells bearing the wild-type p53 gene through apoptosis induction accompanied by activation of caspases, or S-phase arrest by up-regulated cyclin A and B1. ZOL induced p53 phosphorylation and subsequent activation of the downstream pathways. Down-regulated p53 expression with the small interfering RNA, however, showed that both apoptosis and S-phase arrest were irrelevant to the p53 activation. Geranylgeranyl but not farnesyl pyrophosphate inhibited ZOL-induced apoptosis and S-phase arrest, and the geranylgeraniol supplement decreased ZOL-mediated Rap1A but not Ras unprenylation. Inhibition of ERK1/2 pathways suppressed ZOL-induced apoptosis but not S-phase arrest. We further demonstrated that ZOL, administrated intrapleurally, inhibited the tumor growth in the pleural cavity.

Conclusions: These data indicate that ZOL induces apoptosis or S-phase arrest, both of which are independent of p53 activation and Ras unprenylation, and suggest that ZOL is a possible therapeutic agent to mesothelioma partly through non-Ras- and ERK1/2-mediated pathways.

In plain English

This was a laboratory study of a drug, not a study of people or of asbestos exposure. The authors tested zoledronic acid (ZOL), a bisphosphonate bone drug, on eight human mesothelioma cell lines and one immortalized mesothelial cell line. They also tested it in nude mice with mesothelioma cells injected into the pleural (chest) cavity. In the dish, ZOL lowered the readings of a cell-viability test in all lines; the concentration that cut the reading by half (IC50) was under 10 μM. Some cells died by programmed cell death (apoptosis); others stalled in S phase, the DNA-copying phase of the cell cycle. These effects still occurred when p53 levels were lowered with a gene-silencing tool (siRNA) or when p53 was mutated, and geranylgeraniol, but not farnesol, blocked them. Blocking ERK1/2 signaling reduced cell death but not the stall. In mice, ZOL put into the chest cavity reduced tumor weight compared with saline, less so when given later. The authors concluded that intrapleural ZOL could be a new mesothelioma treatment. They noted possible side effects as a concern.

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 study: experiments on cultured cell lines (in vitro) plus an animal experiment in mice (orthotopic model, with tumor cells placed in the pleural cavity)
• Who was studied: Eight human mesothelioma cell lines: MSTO-211H, NCI-H28, NCI-H226, NCI-H2052, NCI-H2452, JMN-1B, EHMES-1 and EHMES-10. Also the immortalized mesothelial cell line Met-5A, and female 6-week-old BALB/c nu/nu (nude) mice. Groups of mice: n = 6 or 7 for the day-3 treatment; for the day-10 treatment, ZOL n = 8 and PBS n = 7.
• Exposure: Treatment with zoledronic acid (ZOL), a third-generation bisphosphonate. Cells received ZOL alone or with farnesol, geranylgeraniol or the MEK1/2 inhibitor PD98059. Mice received 40 μg ZOL, or phosphate buffered saline (PBS) as control, into the pleural cavity on day 3 or day 10. No asbestos or fiber exposure was studied.
• Main results:
  • In all human mesothelioma cell lines tested and in Met-5A cells, the IC50 values of ZOL were less than 10 μM. IC50 here is the concentration that halved the relative reading of the WST viability assay. ZOL decreased numbers of MSTO-211H cells and suppressed proliferation rates of EHMES-10 cells.
  • In MSTO-211H cells, ZOL increased the sub-G1 (dying-cell) population without S-phase arrest. In EHMES-10 cells, ZOL instead increased the S-phase fraction.
  • In EHMES-10 cells, ZOL raised cyclin A and B1, which are markers of S-phase arrest.
  • JMN-1B and EHMES-1 cells carry mutated p53. They were as sensitive to ZOL as the mesothelioma cells with wild-type p53.
  • p53-siRNA suppressed p53 expression levels and inhibited p53 phosphorylation, but did not change ZOL's effects on the cell cycle. The authors concluded the effects were independent of p53 activation.
  • In MSTO-211H and EHMES-10 cells, geranylgeraniol (GGOH) blocked ZOL's reduction of cell viability; farnesol (FOH) did not.
  • The ERK1/2-pathway blocker PD98059 inhibited the ZOL-induced increase in sub-G1 cells in MSTO-211H cells. It did not affect the S-phase arrest in EHMES-10 cells.
  • In mice injected with MSTO-211H or EHMES-10 cells, 40 μg ZOL given into the pleural cavity on day 3 reduced tumor growth compared with the PBS group. The authors reported the effect as dose-dependent.
  • Giving ZOL later, on day 10 after tumors had formed (ZOL n = 8, PBS n = 7), gave a weaker antitumor effect, but tumor growth was still inhibited compared with PBS.
  • A 15 μg ZOL dose given systemically did not slow MSTO-211H tumors growing under the skin (data not shown).
  • In mice without tumors, 40 μg ZOL injected into the pleural cavity caused no pleural effusion and no slowing of body weight gain (data not shown).
• The authors' conclusion: The authors concluded that ZOL was cytotoxic to mesothelioma cells by inducing apoptosis or S-phase arrest, both linked with inhibition of geranylgeranylation. They concluded that ZOL given into the pleural cavity can be a novel therapy for mesothelioma.
• Limitations the authors state:
  • The Met-5A mesothelial cells were already immortalized by SV40 T antigen, which inactivates p53. So it is unclear whether normal mesothelium-derived cells are susceptible to ZOL.
  • The authors failed to detect ZOL-induced apoptotic cells in the mice.
  • Why ZOL causes apoptosis in some cells and S-phase arrest in others remains to be investigated.
  • Possible adverse reactions from giving the drug into the pleural cavity are a concern for clinical use.

Topics

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

Medical terms in this study, explained in our Lexicon: Mesothelioma, Apoptosis, Pleural Cavity.

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