Raltitrexed Inhibits HepG2 Cell Proliferation via G0/G1 Cell Cycle Arrest

Our plain-English summary of Raltitrexed Inhibits HepG2 Cell Proliferation via G0/G1 Cell Cycle Arrest, by H. Zhao, Y. Zhang, J. Sun, et al (Oncology Research, 2016).

The study

• Authors: H. Zhao, Y. Zhang, J. Sun, C. Zhan, L. Zhao
• Journal: Oncology Research
• Published: 2016;23(5):237-48
• Cited by: 25 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Raltitrexed (RTX) is an antimetabolite drug used as a chemotherapeutic agent for treating colorectal cancer, malignant mesothelioma, and gastric cancer. The antitumor capacity of RTX is attributed to its inhibitory activity on thymidylate synthase (TS), a key enzyme in the synthesis of DNA precursors. The current study is aimed at investigating the potential antitumor effects of RTX in liver cancer. Using the HepG2 cell line as an in vitro model of liver cancer, we evaluated the effects of RTX on cell proliferation employing both a WST-8 assay and a clone formation efficiency assay. In addition, we monitored the ultrastructure changes of HepG2 cells in response to RTX with transmission electric microscopy. To investigate the mechanism underlying the regulation of cell proliferation by RTX, we analyzed cell cycle using cell flow cytometry. Moreover, real-time PCR and Western blot analyses were conducted to examine expression levels of cell cycle regulatory proteins cyclin A and cyclin-dependent kinase 2 (CDK2), as well as their mediators tumor suppressor genes p53 and p16. Our results demonstrate that RTX inhibits HepG2 proliferation by arresting the cell cycle at G0/G1. This cell cycle arrest function was mediated via downregulation of cyclin A and CDK2. The observed elevated expression of p53 and p16 by RTX may contribute to the reduction of cyclin A/CDK2. Our study indicates that RTX could serve as a potential chemotherapeutic agent in the treatment of hepatocellular carcinoma.

In plain English

This was a laboratory study of one line of human liver cancer cells (HepG2) grown in dishes. It did not study asbestos exposure or people. The authors tested raltitrexed, a cancer drug they note is used for colorectal cancer, malignant mesothelioma and gastric cancer, at 16, 64 and 256 nM, with another drug, 5-fluorouracil, as a comparison. After 24 hours, raltitrexed cut cell growth by 16.3%, 22.1% and 24.5% at the three doses. After 48 hours the cuts were 33.2%, 34.3% and 36.1%. At 16 nM, treated cells formed 40% fewer colonies than untreated cells. After 24 hours, the share of cells in the combined G0/G1 phases of the cell cycle rose from 60.5% of untreated cells to 86.7% at the highest dose, which the authors interpreted as cell-cycle arrest. Raltitrexed also lowered two proteins that drive cell division, cyclin A and CDK2, and raised two tumor-suppressor proteins, p53 and p16. The authors concluded that raltitrexed could serve as a potential chemotherapy drug for hepatocellular carcinoma, the main type of liver cancer.

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 experiment
• Who was studied: HepG2 human liver carcinoma cells, obtained from Harbin Medical University Cancer Institute, China; no human or animal subjects
• Exposure: The cancer drug raltitrexed (RTX) at 0, 16, 64 and 256 nM for 24 or 48 hours, with 5-fluorouracil (5-FU), another thymidylate synthase inhibitor, as a positive control. No asbestos or fiber exposure was studied.
• Main results:
  • After 24 hours, RTX at 16, 64 and 256 nM inhibited HepG2 cell growth by 16.3 ± 0.5%, 22.1 ± 0.2% and 24.5 ± 0.2%, respectively.
  • After 48 hours, RTX at 16, 64 and 256 nM inhibited HepG2 cell growth by 33.2 ± 0.3%, 34.3 ± 0.9% and 36.1 ± 0.4%.
  • With 24-hour treatment, the IC50 (concentration giving 50% inhibition) for RTX was 78.919 nM.
  • Cells treated with 16 nM RTX formed 40% fewer colonies than control cells (p less than 0.05).
  • Untreated cells were 60.5% in G0/G1, 35.2% in S and 4.3% in G2/M phase. After 24 hours of RTX at 16, 64 and 256 nM, the S-phase share fell to 28.9%, 23.9% and 13.4% (p less than 0.05 vs. control), and the G0/G1 share rose to 71.5%, 76.1% and 86.7%.
  • RTX did not significantly change levels of its target enzyme, thymidylate synthase (TS), in HepG2 cells.
  • At 256 nM, RTX reduced CDK2 protein by 50% compared with control, and CDK2 mRNA fell to 19.0% of control. Cyclin A mRNA and protein were also significantly reduced.
  • RTX raised p53 mRNA by 1.3- up to 5.8-fold compared with control cells, and increased p16 protein and mRNA levels.
  • Under electron microscopy, RTX-treated cells showed damage such as plasma membrane interruption and vacuoles. Early apoptotic cells were seen, but no dividing (mitotic) cells.
• The authors' conclusion: The authors concluded that RTX inhibited HepG2 proliferation by arresting the cell cycle at G0/G1 through downregulation of cyclin A and CDK2. They proposed that increased p53 and p16 may contribute to this, and that RTX may serve as a potential chemotherapeutic agent for hepatocellular carcinoma.

Topics

Medical terms in this study, explained in our Lexicon: Raltitrexed, Colorectal Cancer, Malignant Mesothelioma, Stomach Cancer, Tumor Suppressor Gene, Carcinoma.

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