Serine hydroxymethyltransferase (SHMT) reversibly catalyzes the conversion of serine to glycine. Vertebrates can synthesize glycine in the liver, and the amino acid is a precursor for synthesizing proteins, purines and glutathione. Glycine is important as Li et al. referenced earlier work where cancer cell lines were shown to consume extracellular glycine while normal cells produced glycine. This suggested that cancer cells needed more glycine to grow. Furthermore, AMPA inhibited a biosynthesis pathway that required either of two SHMT isoforms, SHMT1 and SHMT2α (reference 9 cited by Li et al.).
Based on these observations, Li and his colleagues selected eight cancer cell lines (human prostate cancer C4-2B, LNCaP, DU-145 and PC-3; human ovarian cancer SKOV-3 and OVCAR-3; human cervical cancer HeLa and human lung cancer A549) and two normal cell lines (human prostate epithelial RWPE-1 and pRNS-1-1), and treated them with glyphosate or AMPA at three different concentrations for 72 hours to see if there was an effect on cell viability. Using the CellTiter-Glo® Luminescent Cell Viability Assay, which measures the amount of ATP present in the cell, the researchers discovered there was little difference between untreated and glyphosate-treated normal cell lines even at 50mM concentration. However, all cancer cell lines showed reduced proliferation in the presence of 50mM glyphosate albeit some demonstrated more growth inhibition than others (e.g., C4-2B was strongly inhibited while A549 was more modestly inhibited). Glyphosate inhibited cell growth even at lower concentrations with more than half of the cancer cell lines losing some viability when treated with 25mM glyphosate. Similarly, 50mM AMPA inhibited all eight cancer cell lines and left the two normal cell lines unaffected. AMPA seemed to have a slightly stronger effect on the cancer cell lines, as there was reduced growth in all eight cancer cell lines treated with 25mM AMPA. The researchers did test a higher concentration of AMPA (100mM), but even the normal cells showed signs of reduced proliferation.
Two of the cell lines most strongly affected by 50mM AMPA treatment, C4-2B and PC-3, were further investigated. Examining the cells over 24, 48 and 72 hours, Li et al. found that as time increased, more cells became apoptotic, confirming cells were dying, and more died the longer they were exposed to AMPA. Researchers also compared treated and untreated cells to see if there was any change in the cell cycle. Entry into the synthesis (S) phase was inhibited for both C4-2B and PC-3 in the presence of 50mM AMPA for 24 hours. This supported the cell viability and apoptotic data: Fewer of the cells were replicating.
To better understand which cell cycle and apoptosis-related proteins might be affected by AMPA, C4-2B cells were treated with 50mM AMPA for 0, 12, 24, 48 and 72 hours, lysed and analyzed using Western blotting. The authors examined poly(ADP-ribose) polymerase (PARP), a protein that is involved in DNA repair and cleaved during apoptosis. The cleaved form of PARP was present after 12 hours of AMPA treatment. Expression levels of procaspase-9 increased while procaspase-3 decreased over the treatment period. These data also support the earlier apoptosis data as both of these enzymes play a role in programmed cell death. Expression levels of cell cycle proteins tested also were altered. Expression of cyclin D3, a protein involved in progress of the cell cycle, decreased over the tested time while p53 and p21, two proteins that when activated lead to cell cycle arrest, were briefly increased.
This research explored the use of glycine analogs in inhibiting cancer cell line proliferation. Interestingly, researchers demonstrated that normal cultured cells continued to grow when treated with 50mM glyphosate or AMPA while growth of eight commonly used cancer cell lines was negatively affected by extracellular glycine analogs. This work is a preliminary proof of concept and will need further testing in more cancer cell lines as well as animal model systems to confirm that this approach is a useful one. However, if glycine analogs really are effective in organisms, they offer a pathway to explore more effective drugs for cancer therapy.
Li, Q., Lambrechts, M.J., Zhang, Q., Liu, S., Ge, D., Yin, R., Xi, M. and You, Z. (2013) Glyphosate and AMPA inhibit cancer cell growth through inhibiting intracellular glycine synthesis., Drug Design, Development and Therapy, PMID: 23983455
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