THE JOURNAL OF NUTRITION に掲載された論文
The Effects of Branched-Chain Amino Acidson Canine Neoplastic Cell Proliferation and Death
アルギニンの腫瘍細胞増殖抑制作用はin vivoとin vitroの両方で研究されている。
ロイシン処理をした骨肉腫細胞は、細胞周期のS期で停止しG 2 期、M期の喪失を示した。
The Effects of Branched-Chain Amino Acids on Canine Neoplastic Cell Proliferation and Death
The antiproliferative effects of certain amino acids, particularly arginine, have been studied both in vivo and in vitro. Arginine has been used in critical care medicine for some time, and special formulations of arginine-enriched diets are presently being used in human and veterinary medicine. The use of branched-chain amino acids as a therapeutic option for the preservation of lean body mass is becoming increasingly popular. Physicians sometimes advocate their use in cancer-related cachexia; thus, it is increasingly important to understand how high concentrations of these amino acids will affect proliferating neoplastic cells. Regarding cell proliferation, our experiments showed that all amino acids tested had antiproliferative capacities at the highest concentrations (100 mmol/L) but that arginine and leucine were far more suppressive at lower concentrations (50 and 10 mmol/L) than valine or isoleucine.
The antiproliferative effects of all amino acids tested were seen only at extremely high levels. We speculate that this may be because of amino acid imbalance in the medium, considering that all BCAAs compete for the same transporters in the plasma membrane. This amino acid imbalance may cause problems with protein synthesis leading to suppression of growth. The potency of arginine and leucine at inducing cell death suggests that other mechanisms may be playing a role. Thus, we examined whether this was an apoptotic event. Hoechst staining and Western blotting for caspase activity showed caspase-mediated apoptotic events with both arginine and leucine, whereas valine and isoleucine, although adequate at diminishing proliferation, could not induce apoptosis.
In an attempt to understand this cytotoxic response, cell cycle analysis was performed with flow cytometry, which showed a profound difference between arginine and leucine treatment in the osteosarcoma cell line. Leucine-treated cells showed a loss of the G2M phase of the cell cycle with more cells halted in the S phase (Fig. 3). This phenomenon was not seen during the apoptotic events associated with arginine.
To further elucidate the mechanisms underlying the cytotoxic response, a time course analysis was performed in the osteosarcoma cells with arginine and leucine treatments. The events of apoptosis are often characterized by various caspase cascades being activated, all of which converge onto caspase 3, the major caspase activated during induction of apoptosis. The mitochondrial pathways of apoptosis are complex but often result in the activation of caspase 2, 9, or 7 and eventually activation of caspase 3. Caspase 3 then amplifies caspase activation and irreversibly induces apoptosis. Other growth factor–mediated signaling events can led to caspase 3 activation, which is often mediated through activation of caspase 8 or 10.
We used activated caspase 3 as a universal indication of apoptosis, which was activated in the osteosarcoma with both arginine and leucine. The activation occurred within 24 h in arginine-treated cells and within 36 h in the leucine-treated cells. To help determine whether this was a mitochondrial event or through extracellular stimulation, immunoblotting for caspase 2 and caspase 8 was performed. Results showed that neither arginine nor leucine activate caspase 8 and that caspase 2 activation preceded caspase 3 activation; thus, apoptosis caused by mitochondrial dysfunction is likely.
To further identify how leucine was involved in S phase arrest, the inhibitory phosphorylation site on cdc2 (tyrosine 15), which prevents progression through the G2M by preventing cdc2/cyclinB complex from initiating mitosis, was examined through immunoblotting. Cdc2, when bound to cyclin B2, serves as a complex that regulates entry into the G2M phase and changes its phosphorylation state during S phase arrest. As expected, more phosphorylation of tyrosine 15 on cdc2 in the leucine-treated cells was observed, but it was no different from arginine treatment, which also showed excessive phosphorylation. In fact, it appears as if both arginine and leucine activate this mechanism to slow cell proliferation.
Although, ultimately, both amino acid insults result in mitochondrial compromise leading to apoptosis at high concentrations, the differences observed in cell cycle analysis imply that there may be different cytotoxic mechanisms. Further investigation is needed to examine both normal cells and other amino acid treatments on cell proliferation, but our data suggest that BCAA treatment does not directly potentiate neoplastic cell growth and may actually diminish neoplastic cell proliferation at supraphysiological concentrations.