Nitric oxide (NO) enhances pemetrexed cytotoxicity via NO‑cGMP signaling in lung adenocarcinoma cells in vitro and in vivo

Our plain-English summary of Nitric oxide (NO) enhances pemetrexed cytotoxicity via NO‑cGMP signaling in lung adenocarcinoma cells in vitro and in vivo, by H. Nagai, H. Yasuda, Y. Hatachi, et al (International Journal of Oncology, 2012).

The study

• Authors: H. Nagai, H. Yasuda, Y. Hatachi, D. Xue, T. Sasaki, M. Yamaya, Y. Sakamori, Y. Togashi, K. Masago, I. Ito, Y.H. Kim, T. Mio, M. Mishima
• Journal: International Journal of Oncology
• Published: 2012;41(1):24-30
• Cited by: 23 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

Pemetrexed (PEM) is a novel, multitargeted, antifolate, antineoplastic agent for the treatment of non-small cell lung cancer and malignant pleural mesothelioma. Additional effects of nitric oxide (NO) donors on the chemosensitivity of cancers have been reported. However, the effects of an NO donor on PEM-induced cytotoxicity remain unknown. In this study, we investigated the effects of the NO donors, NOC-18 on the cytotoxicity in A549 cells in vitro and of nitroglycerin (GTN), on the tumor growth of Lewis lung carcinoma cells in a murine syngraft model treated with PEM. The effects of NO donors on the expression of proteins associated with PEM metabolism, including thymidylate synthase (TS), reduced folate carrier 1 (RFC1), folylpolyglutamate synthase (FPGS), γ-glutamyl hydrolase (GGH) and multidrug resistance-related protein (MRP)5, and the effects of cyclic guanosine mono-phosphate (cGMP) signaling on these proteins were examined in A549 cells. Treatment with 100 nM NOC-18 for 3 days significantly enhanced PEM-induced cytotoxicity and increased the expression of RFC1 and FPGS in A549 cells. Treatment with 10 nM 8-bromo-cGMP (8-Br-cGMP) for 3 days also increased the expression of RFC1 and FPGS in A549 cells. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (10 µm) significantly reversed the increase in RFC1 and FPGS expression induced by 100 nM NOC-18 in A549 cells. Combination therapy with GTN and PEM significantly reduced tumor growth compared with PEM alone in the syngraft model. The enhanced antitumor effect of GTN plus PEM was significantly reversed by the concomitant addition of ODQ. These findings suggest that NO donors, such as NOC-18 and GTN, enhance the anticancer effects of PEM by increasing the RFC1 and FPGS expression and stimulating cGMP signaling pathways in cancer cells.

In plain English

This laboratory study from Japan did not examine asbestos exposure or people with mesothelioma. It tested whether nitric oxide (NO) donor drugs changed the effect of pemetrexed, a chemotherapy drug the authors describe as used for non-small cell lung cancer and malignant pleural mesothelioma. Human lung adenocarcinoma cells (A549) were grown in low-oxygen conditions. In these cells, 3 days of pretreatment with 100 nM of the NO donor NOC-18, followed by pemetrexed, significantly increased cell death compared with pemetrexed alone. With a 10 nM dose, the authors found no statistically significant increase. The 100 nM dose also raised levels of two proteins involved in pemetrexed uptake and retention, RFC1 and FPGS. A blocker of cGMP signaling (ODQ) reversed this rise. In mice with implanted Lewis lung carcinoma tumors (seven per group), nitroglycerin plus pemetrexed significantly reduced tumor growth at day 15 compared with pemetrexed alone. Nitroglycerin alone did not affect tumor growth. The authors concluded that NO donors enhance pemetrexed's effect through NO-cGMP signaling, and said this should be verified in patients. They noted that they did not measure pemetrexed levels inside the cells.

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: cell-culture (in vitro) experiments and an animal experiment in mice (syngraft tumor model)
• Who was studied: Human lung adenocarcinoma cells (A549) cultured under hypoxic conditions (1% oxygen). Murine Lewis lung carcinoma (LLC) cells injected under the skin of 6-week-old female NCr C57/BL6 mice, which were randomly allocated to 5 treatment groups of seven mice each. No human subjects and no asbestos-exposed population.
• Exposure: No asbestos exposure was studied. The exposures were drugs. In cells: the NO donor NOC-18 (10 or 100 nM for 3 days), then pemetrexed (200 µM for 3 h); also 8-Br-cGMP (10 nM) and the guanylate cyclase inhibitor ODQ (10 µM). In mice: nitroglycerin (GTN) 0.2 mg/kg, pemetrexed 50 mg/kg and ODQ 10 mg/kg, given by intraperitoneal injection for 2 weekly courses.
• Main results:
  • In A549 cells, pemetrexed combined with 10 nM NOC-18 showed no statistically significant increase in apoptosis compared with pemetrexed alone. Pemetrexed combined with 100 nM NOC-18 showed a statistically significant increase compared with pemetrexed alone or with the lower dose (p<0.01). Results were shown in figures, with no numeric values given in the text.
  • In A549 cells, 100 nM NOC-18 significantly increased RFC1 and FPGS mRNA (p<0.01) and increased the corresponding proteins compared with controls. There were no differences for TS, GGH and MRP5.
  • In A549 cells, 10 nM 8-Br-cGMP significantly upregulated RFC1 and FPGS mRNA (p<0.01). Adding 10 µM ODQ to 100 nM NOC-18 significantly decreased RFC1 and FPGS mRNA compared with NOC-18 alone (p<0.01).
  • In mice with LLC tumors, nitroglycerin alone did not affect tumor growth compared with control. Pemetrexed alone attenuated growth compared with control at day 15 (p<0.05). Pemetrexed plus nitroglycerin significantly decreased tumor growth compared with pemetrexed alone at day 15 (p<0.05). Adding ODQ significantly increased tumor growth compared with pemetrexed plus nitroglycerin.
• The authors' conclusion: The authors concluded that NO donors enhance the cytotoxicity of pemetrexed via NO-cGMP signaling and an increase in RFC1 and FPGS expression in lung adenocarcinoma cells. They added that NO donors such as nitroglycerin may be promising agents, and that the combination should be verified in a clinical setting.
• Limitations the authors state:
  • The authors did not measure the actual concentration of pemetrexed inside the cells, so the effect of NOC-18 on intracellular pemetrexed remains unclear.
  • NOC-18 alone increased apoptosis in cells, but nitroglycerin alone did not reduce tumor growth in mice. The authors attributed this partly to differences between cell-culture and animal settings and said further studies are needed on NO donors used alone.

Topics

Medical terms in this study, explained in our Lexicon: Pemetrexed (Alimta), Lung Adenocarcinoma, Antifolate, Non-Small Cell Lung Cancer, Pleural Mesothelioma, A549 Cells, Carcinoma.

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